SYSTEMIC DELIVERY OF MYOSTATIN SHORT INTERFERING NUCLEIC ACIDS (siNA) CONJUGATED TO A LIPOPHILIC MOIETY

ABSTRACT

The present invention provides methods comprising the in vivo delivery of small nucleic acid molecules capable of mediating RNA interference and reducing the expression of myostatin, wherein the small nucleic acid molecules are introduced to a subject by systemic administration. Specifically, the invention relates to methods comprising the in vivo delivery of short interfering nucleic acid (siNA) molecules that target a myostatin gene expressed by a subject, wherein the siNA molecule is conjugated to a lipophilic moiety, such as cholesterol. The myostatin siNA conjugates that are delivered as per the methods disclosed are useful to modulate the in vivo expression of myostatin, increase muscle mass and/or enhance muscle performance. Use of the disclosed methods is further indicated for treating musculoskeletal diseases or disorders and/or diseases or disorders that result in conditions in which muscle is adversely affected.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/902,358, filed Nov. 11, 2013, the entire contents of which areincorporated herein by reference.

REFERENCE TO SEQUENCE LISTING

A sequence listing text file is submitted via EFS-Web in compliance with37 CFR §1.52(e)(5) concurrently with the specification. The sequencelisting has the file name “A2038-7219WO Sequence Listing”, was createdon Nov. 10, 2014, and is 24,773 bytes in size. The sequence listing ispart of the specification and is incorporated in its entirety byreference herein.

BACKGROUND OF THE INVENTION

RNA interference (RNAi) is an evolutionarily conserved cellularmechanism of post-transcriptional gene silencing found in fungi, plantsand animals that uses small RNA molecules to inhibit gene expression ina sequence-specific manner. RNAi is controlled by the RNA-inducedsilencing complex (RISC) that is initiated by short double-stranded RNAmolecules in a cell's cytoplasm. The short double-stranded RNA interactswith Argonaute 2 (Ago2), the catalytic component of RISC, which cleavestarget mRNA that is complementary to the bound RNA. One of the two RNAstrands, known as the guide strand, binds the Ago2 protein and directsgene silencing, while the other strand, known as the passenger strand,is degraded during RISC activation. See, for example, Zamore and Haley,2005, Science, 309:1519-1524; Vaughn and Martienssen, 2005, Science,309:1525-1526; Zamore et al., 2000, Cell, 101:25-33; Bass, 2001, Nature,411:428-429; and, Elbashir et al., 2001, Nature, 411:494-498.Single-stranded short interfering RNA has also been shown to bind Ago2and support cleavage activity (see, e.g., Lima et al., 2012, Cell150:883-894).

The RNAi machinery can be harnessed to destroy any mRNA of a knownsequence. This allows for suppression (knockdown) of any gene from whichit was generated, consequently preventing the synthesis of the targetprotein. Modulation of gene expression through an RNAi mechanism can beused to modulate therapeutically relevant biochemical pathways,including ones which are not accessible through traditional smallmolecule control.

Chemical modification of nucleotides incorporated into RNAi moleculesleads to improved physical and biological properties, such as nucleasestability (see, e.g., Damha et al., 2008, Drug Discovery Today,13:842-855), reduced immune stimulation (see, e.g., Sioud, 2006, TRENDSin Molecular Medicine, 12:167-176), enhanced binding (see, e.g., Koller,E. et al., 2006, Nucleic Acid Research, 34:4467-4476), and enhancedlipophilic character to improve cellular uptake and delivery to thecytoplasm. Thus, chemical modifications have the potential to increasepotency of RNA compounds, allowing lower doses of administration,reducing the potential for toxicity, and decreasing overall cost oftherapy.

In recent years, advances in oligonucleotide design and chemicalmodification types/patterns have resulted in molecules with increasedresistance to nuclease-mediated degradation, improved pharmokinetics,increased gene specificity and reduced immunostimulatory responses(Lares, M. R. et al. 2010, Trends Biotechnol. 58:570-9). Despite thesemajor advances, siRNA delivery to a diverse range of tissues remains amajor obstacle in vivo. While siRNA delivery in vivo has been achievedin eye, lung, brain, tumor, and muscle by localized delivery (byintraocular, intranasal, intrathecal, intratumoral, and intramuscularinjections, respectively), this delivery method is only suitable fortarget validation studies due to its invasive nature and has limitedrelevance as a clinical therapy (Golzio, M. et al., 2005, Gene Ther.12:246-51; Liang, Y. et al., 2010, PLoS One 5:e12860; Reich, S. J. etal., 2003, Mol. Vis. 9:210-6; Tan, P. H. et al., 2005, Gene Ther.12:59-66; Zhang, X. et al., 2004, J. Biol. Chem. 279:10677-84). A goodsystemic delivery system is essential to reach certain tissues ofinterest. Numerous studies have demonstrated systemic and targetedsystemic siRNA delivery in vivo through a variety of methods, includingcationic lipid and polymers, cholesterol conjugates, cell-penetratingpeptides, recombinant viral vectors, small molecule carriers,antibody-linked siRNA and targeting ligands (Frank-Kamenetsky, M. etal., 2008, Proc. Natl. Acad. Sci. USA 105:11915-20; Khoury, M. et al.,2006, Arthritis Rheum. 54:1867-77; Kim, B. et al., 2004, Am. J. Pathol.165:2177-85; Kondo, E. et al., 2012, Nat. Commun. 3:951; Morrissey, D.V. et al., 2005, Nat. Biotechnol. 23:1002-7; Schiffelers, R. M. et al.,2004, Nucleic Acids Res. 32:e149; Song, E. et al., 2005, Nat.Biotechnol. 23:709-17; Wolfrum, C. S. et al., 2007, Nat. Biotechnol.25:1149-57). However, systemic siRNA delivery has remained limited toparticular tissues, such as liver, tumors, spleen and jejunum (Abrams,M. T. et al., 2010, Mol. Ther. 18:171-80; Chien, P. Y. et al., 2005,Cancer Gene Ther. 12:321-8; Liang, Y. et al., supra; Sorensen, D. R. etal., 2003, J. Mol. Biol. 327:761-6; Tadin-Strapps, M. et al., 2011, J.Lipid Res. 52:1084-97; Wolfrum, C. et al., supra).

Myostatin is an inhibitor of skeletal muscle differentiation and growth.During development it is an inhibitor of myogenesis, while duringadulthood its major role is in negatively regulating satellite cellactivation and self-renewal. Myostatin is a member of the TGF-β familyand acts as a catabolic stimulus through the ActRIIB receptor to induceSMAD2/3/FOXO/NF-κB signaling and muscle fiber atrophy (Sartori, R. G. etal., 2009, Am. J. Physiol. Cell Physiol. 296:C1248-57; Stitt, T. N. etal., 2004, Mol. Cell 14:395-403). Myostatin knockout mice, as well asother mouse models of myostatin inhibition, display increased musclemass/strength and an attenuated/reversal of a muscle atrophy phenotypein different muscle disease models (Akpan, I. et al., 2009, Int. J.Obes. (Lond) 33:1265-73; Heineke, J. et al., 2010, Circulation121:419-25; Lin, J. et al., 2002, Biochem. Biophys. Res. Commun.291:701-6; Zhang, L. 2011, Faseb J. 25:1653-63; Zhou, X. et al., 2010,Cell 142:531-43). Small-interfering RNAs targeting myostatin may havenumerous therapeutic applications in the multitude of existing muscledisorders, which range from muscular dystrophy, muscular atrophy incachexia-inducing diseases, such as cancer, heart disease, chronicobstructive pulmonary disease, sarcopenia, chronic kidney disease, andmetabolic diseases, and also in insulin-resistant disorders (Asp, M. L.et al., 2010, Int. J. Cancer 126:756-63; Bailey, J. L. et al., 2006, J.Am. Soc. Nephrol. 17:1388-94; Engelen, M. P. et al., 1994, Eur. Respir.J. 7:1793-7; Ruegg, M. A. et al., 2011, Annu. Rev. Pharmacol. Toxicol.51:373-95).

To date there has been limited success in siRNA or antisenseoligonucleotide (ASO) delivery systemically to muscle, with most reportshighlighting muscle targeting by local injection (Gebski, B. L. et al.,2003, Hum. Mol. Genet. 12:1801-11; Guess, M. G. et al., 2013, Skelet.Muscle 3:19; Laws, N. et al., 2008, J. Appl. Physiol. 105:662-8; Tang,Y. et al., 2012, Mol. Pharmacol. 82:322-32). Several studies have usedelectroporation additively with intramuscular (IM) injections to improvethe transfer of siRNAs or plasmid vectors into muscle cells (Eefting, D.et al., 2007, Hum. Gene Ther. 18:861-9; Golzio, M. et al., 2005, supra;Kishida, T. et al., 2004, J. Gene Med. 6:105-10). However, IM injectionshave a long-standing history for causing pain, local muscle damage andinflammation, which also minimizes their usefulness for therapeuticapplications (McMahon, J. M. et al., 1998, Gene Ther. 5:1283-90). As animprovement to IM delivery, a model of “local” venous delivery musclesystem was developed, which involves the use of a tourniquet totransiently isolate the injection solution in the muscle of the limb, inorder to deliver a “high pressure” hydrodynamic injection of aluciferase pDNA vector to muscle in rats, dogs and monkeys (Hagstrom, J.E. et al., 2004, Mol. Ther. 10:386-98). Although it showed successfuldelivery into multiple muscle groups in the limb and the ability formultiple dosing, delivery efficiency was low and it is still an invasivetechnique that requires a high degree of injection skill.

In recent years, the use of the carrier polymer, atelocollagen, has beenused for delivery of nucleic acids (siRNA, ASOs and plasmids) andnegatively-charged proteins. Recent studies shows both local andsystemic delivery of an atelocollagen/siRNA complex to muscle in a modelof Duchenne muscular dystrophy (DMD) (Kawakami, E. et al., 2013, PLoSOne 8:e64719; Kawakami, E. et al., 2011, Dev. Growth Differ. 53:48-54;Kinouchi, N. et al., 2008, Gene Ther. 15:1126-30).

There continues to be a need to develop therapies that can easily andnon-invasively deliver nucleic acids to the muscle, which could have thepotential for use in the future treatment of a variety of muscledisorders, such as muscular atrophic diseases, muscular dystrophy, andtype II diabetes.

SUMMARY OF THE INVENTION

The present invention provides methods for delivering to a subject smallnucleic acid molecules capable of mediating RNA interference andreducing the expression of myostatin. The small nucleic acid moleculesof the invention are more specifically referred to herein as shortinterfering nucleic acid (siNA) molecules. The siNA molecules that aredelivered as per the methods disclosed target a myostatin gene and areconjugated to a lipophilic moiety, such as cholesterol (i.e., myostatinsiNA conjugates). Once delivered to their site of action (e.g., musclecells that express myostatin), the myostatin siNA conjugates act toinhibit or down regulate myostatin gene expression by causingdestruction of a myostatin gene. By reducing the expression of amyostatin gene and, in turn, reducing the level of myostatin protein,the methods of the invention have the potential of enhancing muscle massand/or function. Thus, use of the disclosed methods is indicated, forexample, for treating musculoskeletal diseases/disorders anddiseases/disorders that result in conditions in which muscle isadversely affected, such as neurodegenerative diseases/disorders,sarcopenia, cachexia, obesity, Type-II diabetes, HIV/AIDS and cancer.The methods of the invention are also useful, for example, for enhancingmuscle mass and/or function in livestock including, but not limited to,cattle, pigs and fowl.

An embodiment of the present invention relates to methods of deliveringto the muscle of a subject a short interfering nucleic acid (siNA)molecule, or pharmaceutical compositions thereof, that targets amyostatin gene comprising the step of systemically administering to saidsubject a conjugate of said siNA, wherein said conjugate comprises thesiNA molecule linked to a lipophilic moiety (e.g., cholesterol). Thus,the present invention relates to methods of delivering to the muscle ofa subject myostatin siNA conjugates, or pharmaceutical compositionsthereof, via systemic administration. The myostatin siNA conjugates thatare systemically administered to the subject are delivered to musclethat expresses myostatin. Once delivered to the muscle, the myostatinsiNA conjugate reduces myostatin expression by an RNA interferencemechanism. Thus, the present invention relates to methods of deliveringto the muscle of a subject a myostatin siNA conjugate, or apharmaceutical composition thereof, comprising systemicallyadministering the myostatin siNA conjugate to said subject in an amounteffective to modulate (e.g., inhibit or down-regulate) myostatinexpression in said muscle, wherein said myostatin siNA conjugatecomprises an siNA molecule that targets a myostatin gene expressed bysaid subject linked to a lipophilic moiety (e.g., cholesterol), andwherein said myostatin siNA conjugate mediates RNA interference.

An embodiment of the present invention relates to methods of modulating(e.g., inhibiting or down-regulating) in vivo expression of a myostatingene in a subject comprising introducing to said subject an effectiveamount of a myostatin siNA conjugate, or a pharmaceutical compositionthereof, by systemic administration, wherein the siNA conjugatecomprises a siNA molecule that targets a myostatin gene expressed bysaid subject linked to a lipophilic moiety (e.g., cholesterol), andwherein the siNA conjugate mediates RNA interference. Another embodimentrelates to methods of modulating (e.g., inhibiting or down-regulating)in vivo expression of a myostatin gene in a subject comprisingdelivering to the muscle of said subject an effective amount of amyostatin siNA conjugate, or a pharmaceutical composition thereof, byintroducing the siNA conjugate to said subject by systemicadministration, wherein the siNA conjugate comprises a siNA moleculethat targets a myostatin gene expressed by said subject linked to alipophilic moiety (e.g., cholesterol), and wherein the siNA conjugatemediates RNA interference. Thus, the present invention relates tomethods of modulating in vivo expression of a myostatin gene in asubject comprising systemically administering an effective amount of ansiNA conjugate, or a pharmaceutical composition thereof, to saidsubject, wherein the siNA conjugate comprises an siNA molecule thattargets a myostatin gene expressed by said subject linked to alipophilic moiety, and wherein said siNA conjugate mediates RNAinterference. The myostatin siNA conjugates that are systemicallyadministered to the subject are delivered to muscle that expressesmyostatin and, by an RNA interference mechanism, inhibits ordown-regulates the expression of a myostatin gene in the muscle.

A further aspect of the invention includes myostatin siNA conjugates foruse to modulate in vivo expression of a myostatin gene expressed by asubject (i.e., a human or animal). Another embodiment relates to the useof a conjugate comprising an siNA molecule that targets a myostatin geneand a lipophilic moiety (e.g., cholesterol), for the manufacture of amedicament for modulating in vivo expression of a myostatin geneexpressed by a subject, which comprises systemic administration of saidconjugate, or a pharmaceutical composition thereof, to said subject.

Another embodiment of the present invention provides methods forenhancing muscle mass in a subject comprising reducing myostatin levelsin said subject by introducing to said subject an effective amount of amyostatin siNA conjugate, or a pharmaceutical composition thereof, bysystemic administration, wherein the siNA conjugate comprises an siNAmolecule that targets a myostatin gene expressed by said subject linkedto a lipophilic moiety (e.g., cholesterol), and wherein the siNAconjugate mediates RNA interference. Another embodiment relates tomethods for enhancing muscle mass in a subject comprising reducingmyostatin levels in said subject by delivering to the muscle of saidsubject an effective amount of a myostatin siNA conjugate, or apharmaceutical composition thereof, by introducing the siNA conjugate tosaid subject by systemic administration, wherein the siNA conjugatecomprises an siNA molecule that targets a myostatin gene expressed bysaid subject linked to a lipophilic moiety (e.g., cholesterol), andwherein the siNA conjugate mediates RNA interference. The phrase“reducing myostatin levels,” as used herein, refers to either reducingexpression of a myostatin gene or reducing myostatin protein levels. Themyostatin siNA conjugates that are systemically administered to thesubject are delivered to muscle that expresses myostatin and, by an RNAinterference mechanism, inhibits or down-regulates the expression of amyostatin gene in the muscle. The decrease in myostatin expressionresults in an increase in the muscle mass of the subject. The terms“muscle enhancement” and “enhancing muscle” are intended to beinterchangeable herein and include, but are not limited to, inducementof hyperplasia (increased muscle fiber number), inducement ofhypertrophy (increased muscle fiber diameter) or both. The increase canbe in type 1 and/or type 2 muscle fibers. This aspect of the inventionfurther relates to methods of regenerating injured musculoskeletaltissue in a subject in need thereof by systemically delivering myostatinsiNA conjugates, or pharmaceutical compositions thereof, describedherein.

A further aspect of the invention includes myostatin siNA conjugates foruse to enhance muscle mass and/or to regenerate injured musculoskeletaltissue in a subject. Another embodiment relates to the use of aconjugate comprising an siNA molecule that targets a myostatin genelinked to a lipophilic moiety (e.g., cholesterol), for the manufactureof a medicament for enhancing muscle mass and/or regenerating injuredmusculoskeletal tissue in a subject, which comprises systemicadministration of said conjugate, or a pharmaceutical compositionthereof, to said subject.

Another embodiment of the present invention provides methods forenhancing muscle performance in a subject comprising reducing myostatinlevels in said subject by introducing to said subject an effectiveamount of a myostatin siNA conjugate, or a pharmaceutical compositionthereof, by systemic administration, wherein the siNA conjugatecomprises an siNA molecule that targets a myostatin gene expressed bysaid subject linked to a lipophilic moiety (e.g., cholesterol), andwherein the siNA conjugate and mediates RNA interference. Anotherembodiment relates to methods for enhancing muscle performance in asubject comprising reducing myostatin levels in said subject bydelivering to the muscle of said subject an effective amount of amyostatin siNA conjugate, or a pharmaceutical composition thereof, byintroducing the siNA conjugate to said subject by systemicadministration, wherein the siNA conjugate comprises an siNA moleculethat targets a myostatin gene expressed by said subject linked to alipophilic moiety (e.g., cholesterol), and wherein the siNA conjugatemediates RNA interference. The myostatin siNA conjugates that aresystemically administered to the subject are delivered to muscle thatexpresses myostatin and, by an RNA interference mechanism, inhibits ordown-regulates the expression of a myostatin gene in the muscle. Thedecrease in myostatin expression results in an increase muscleperformance in the subject. “Enhanced muscle performance” includes, butis not limited to, one or more of decreased atrophy, increased muscleendurance and increased overall muscle strength (e.g., increasedcontractile force). A further aspect of the invention includes myostatinsiNA conjugates for use to enhance muscle performance in a subject.

Another embodiment of the invention relates to methods of treatingmusculoskeletal diseases or disorders and/or diseases or disorders thatresult in conditions in which muscle is adversely affected (e.g., muscleweakness, muscle atrophy) in a subject in need thereof comprising thestep of reducing myostatin levels in said subject by introducing to saidsubject an effective amount of a myostatin siNA conjugate, or apharmaceutical composition thereof, by systemic administration, whereinthe siNA conjugate comprises an siNA molecule that targets a myostatingene expressed by said subject linked to a lipophilic moiety (e.g.,cholesterol), and wherein the siNA conjugate mediates RNA interference.A further embodiment of the invention relates to methods of treatingmusculoskeletal diseases or disorders and/or diseases or disorders thatresult in conditions in which muscle is adversely affected (e.g., muscleweakness, muscle atrophy) in a subject in need thereof comprising thestep of reducing myostatin levels in said subject by delivering to themuscle of said subject an effective amount of a myostatin siNAconjugate, or a pharmaceutical composition thereof, by systemicadministration, wherein the siNA conjugate comprises an siNA moleculethat targets a myostatin gene expressed by said subject linked to alipophilic moiety (e.g., cholesterol), and wherein the siNA conjugatemediates RNA interference. The myostatin siNA conjugates that aresystemically administered to the subject are delivered to muscle thatexpresses myostatin and, by an RNA interference mechanism, inhibits ordown-regulates the expression of a myostatin gene in the muscle. Thedecrease in myostatin expression results in an increased muscle massand/or enhanced muscle performance in the subject.

A further aspect of the invention includes myostatin siNA conjugates foruse to treat musculoskeletal diseases or disorders and/or diseases ordisorders that result in conditions in which muscle is adverselyaffected in a subject. Another embodiment relates to the use of aconjugate comprising an siNA molecule that targets a myostatin genelinked to a lipophilic moiety (e.g., cholesterol), for the manufactureof a medicament for treating musculoskeletal diseases or disordersand/or diseases or disorders that result in conditions in which muscleis adversely affected in a subject, which comprises systemicadministration of said conjugate, or a pharmaceutical compositionthereof, to said subject.

The methods of the present invention can be performed on a subject towhich nucleic acid molecules can be systemically administered. The term“subject” as used herein is intended to include human and non-humananimals. Non-human animals include all vertebrates, for example, mammalsand non-mammals, such as non-human primates, sheep, dogs, cats, cows,horses, chickens, amphibians, and reptiles. In one embodiment, themethods of the present invention are performed on a mammal. In anotherembodiment, the methods of the present invention are performed onlivestock. In another embodiment, the methods of the present inventionare performed on humans. In a further embodiment, the human is diagnosedwith musculoskeletal disease. The term “subject” is also intended toinclude an embryo, including a chicken embryo contained within an egg.

An embodiment of the present invention relates to a conjugate comprisingan siNA molecule that targets a myostatin gene and a lipophilic moiety(e.g., cholesterol). The myostatin siNA conjugates of the invention maybe used in a method of treatment of a subject by therapy, whichcomprises systemic administration of said conjugate, or a pharmaceuticalcomposition thereof, to said subject. A further embodiment relates tothe use of a conjugate comprising an siNA molecule that targets amyostatin gene and a lipophilic moiety (e.g., cholesterol), for themanufacture of a medicament for treating a subject, which comprisessystemic administration of said conjugate, or a pharmaceuticalcomposition thereof, to said subject.

An embodiment of the present invention relates to a conjugate comprisingan siNA molecule that targets a myostatin gene and a lipophilic moiety(e.g., cholesterol), for use in a method of treatment of a subject bytherapy, wherein the conjugate is formulated for systemicadministration. A further embodiment relates to the use of a conjugatecomprising an siNA molecule that targets a myostatin gene and alipophilic moiety (e.g., cholesterol), for the manufacture of amedicament for treating a subject, wherein the conjugate is formulatedfor systemic administration.

The myostatin siNA conjugates of the present invention that aredelivered by the disclosed methods comprise a myostatin siNA moleculelinked to a lipophilic moiety. The myostatin siNA conjugates deliveredby the methods of the present invention are not formulated with lipidformulations that form liposomes. While not wishing to be bound by aparticular theory, it is believed the attachment of a lipophilic moietyto the myostatin siNA molecule increases the lipophilicity of the siNAmolecule, enhancing the entry of the siNA molecule into muscle cells.Examples of lipophilic moieties that can be linked to the myostatin siNAmolecule include, but are not limited to cholesterol, oleic acid,stearic acid, palmitic acid, myristic acid, linoleic acid, oleyl,retinyl, cholesteryl residues, cholic acid, adamantane acetic acid,1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl,or phenoxazine. A preferred lipophilic moiety is cholesterol.

The lipophilic moiety is attached to the myostatin siNA molecule throughlinkage to a terminus of the siNA molecule (e.g., the 3′ or 5′ end ofthe sense strand of the siNA molecule) or through linkage to an internalnucleotide of the siNA molecule. In one embodiment, the lipophilicmoiety is attached to the 3′ end of the passenger strand (sense strand)of a double-stranded myostatin siNA molecule. In one embodiment, thelipophilic moiety is attached to the 5′ end of the passenger strand of adouble-stranded myostatin siNA molecule. In a further embodiment, thelipophilic moiety is attached to the 3′ end of the guide strand(antisense strand) of a myostatin siNA molecule. In a furtherembodiment, a myostatin siNA conjugate contains more than one attachedlipophilic moiety (e.g., a lipophilic moiety attached to both the 3′ andthe 5′ end of the passenger strand; a lipophilic moiety attached to the3′ end of the guide strand and the 5′ end of the passenger strand). Inthis aspect of the invention, the lipophilic moieties can be the same ordifferent.

The present invention further provides siNA molecules useful formodulating the expression of myostatin genes and to which a lipophilicmoiety can be attached to form the myostatin siNA conjugates describedherein. The siNA portion of the myostatin siNA conjugates that aredelivered by the methods of the present invention can be single- ordouble-stranded small interfering nucleic acid molecules and can takedifferent oligonucleotide forms, including but not limited to shortinterfering RNA (siRNA), double-stranded RNA (dsRNA) and short hairpinRNA (shRNA) molecules. In one embodiment, the myostatin siNA moleculesare double-stranded siNA molecules comprising a sense and an antisensestrand. The antisense strand comprises a sequence that is complementaryto a portion of a myostatin target RNA sequence, and the sense strand iscomplementary to at least part of the antisense strand. Thedouble-stranded myostatin siNA molecules delivered by the methods of thepresent invention can be symmetric or asymmetric. In another aspect, themyostatin siNA molecules are single-stranded siNA molecules, wherein thesingle oligonucleotide strand (the antisense strand) comprises asequence that is complementary to at least part of a myostatin targetRNA sequence. The siNA portion of the myostatin siNA conjugates that aredelivered by the methods of the present invention inhibit myostatin geneexpression in a subject via an RNA interference (RNAi) mechanism.

The myostatin siNA conjugates described herein are directed to amyostatin gene that can be derived from any of a number of animalspecies, including but not limited humans, cattle, swine, fowl androdent. In one embodiment, the myostatin gene is a human myostatin RNA.In another embodiment, the myostatin gene is a cattle myostatin RNA. Inanother embodiment, the myostatin gene is a swine myostatin RNA. In afurther embodiment, the myostatin gene is a fowl myostatin RNA (e.g.,chicken, turkey). In a further embodiment, the myostatin gene is arodent myostatin RNA (e.g., mouse).

In certain embodiments, the siNA molecules of the siNA portion of themyostatin siNA conjugates that are delivered by the methods of thepresent invention comprise an antisense strand having at least 15nucleotides with sequence complementarity to a myostatin gene sequence.In other embodiments, the antisense strand of an siNA molecule deliveredby the methods of the present invention is about 15 to 30 nucleotides inlength. In further embodiments, a double-stranded siNA moleculedelivered by the methods of the present invention comprises a sensestrand and an antisense strand, wherein each strand is independentlyabout 15 to 30 nucleotides in length.

In one embodiment, the siNA portion of the siNA conjugates of theinvention are double-stranded siNA molecules that modulate theexpression of a myostatin gene, wherein the siNA molecule comprises asense strand and an antisense strand, wherein each strand isindependently 15 to 30 nucleotides in length, and wherein the antisensestrand comprises at least 15 nucleotides having sequence complementaryto any of:

(SEQ ID NO: 1) 5′- AUGGCAAAGAACAAAUAAU -3′; (SEQ ID NO: 2)5′- GGCAAAGAACAAAUAAUAU -3′; (SEQ ID NO: 3) 5′- ACUCCAGAAUAGAAGCCAU -3′;or (SEQ ID NO: 4) 5′- UUUGGAAGAUGACGAUUAU -3′.In one embodiment, the “at least 15 nucleotides” are 15 contiguousnucleotides.

In some embodiments, the antisense strand of the siNA molecule portionof the myostatin siNA conjugates of the invention comprises at least 15nucleotides having sequence identity to any of:

(SEQ ID NO: 18) 5′- AUUAUUUGUUCUUUGCCAU -3′; (SEQ ID NO: 19)5′- AUAUUAUUUGUUCUUUGCC -3′; (SEQ ID NO: 20)5′- AUGGCUUCUAUUCUGGAGU -3′; or (SEQ ID NO: 21)5′- AUAAUCGUCAUCUUCCAAA -3′.In one embodiment, the “at least 15 nucleotides” are 15 contiguousnucleotides. Thus, the antisense strand of the siNA molecule comprisesat least a 15 nucleotide sequence of any of SEQ ID NOs: 18-21.

In some embodiments, the sense strand of the siNA molecule portion ofthe myostatin siNA conjugates of the invention comprises at least 15nucleotides having sequence identity to any of:

(SEQ ID NO: 1) 5′- AUGGCAAAGAACAAAUAAU -3′; (SEQ ID NO: 2)5′- GGCAAAGAACAAAUAAUAU -3′; (SEQ ID NO: 3) 5′- ACUCCAGAAUAGAAGCCAU -3′;or (SEQ ID NO: 4) 5′- UUUGGAAGAUGACGAUUAU -3′.In one embodiment, the “at least 15 nucleotides” are 15 contiguousnucleotides. Thus, the sense strand of the siNA molecule comprises atleast a 15 nucleotide sequence of any of SEQ ID NOs: 1-4.

In some embodiments, the siNA molecule portion of the myostatin siNAconjugates of the invention comprises at least a 15 nucleotide sequenceof both SEQ ID NO: 1 and 18; or both SEQ ID NO: 2 and 19; or both SEQ IDNO: 3 and 20; or both SEQ ID NO: 4 and 21. In another embodiment, thesiNA molecule portion of the myostatin siNA conjugates comprises any ofthe following double-stranded molecules:

(SEQ ID NO: 1) 5′- AUGGCAAAGAACAAAUAAU -3′ and (SEQ ID NO: 18)5′- AUUAUUUGUUCUUUGCCAU -3′; (SEQ ID NO: 2) 5′- GGCAAAGAACAAAUAAUAU -3′and (SEQ ID NO: 19) 5′- AUAUUAUUUGUUCUUUGCC -3′; (SEQ ID NO: 3)5′- ACUCCAGAAUAGAAGCCAU -3′ and (SEQ ID NO: 20)5′- AUGGCUUCUAUUCUGGAGU -3′; or (SEQ IN NO: 4)5′- UUUGGAAGAUGACGAUUAU -3′ and (SEQ ID NO: 21)5′- AUAAUCGUCAUCUUCCAAA -3′.

In some embodiments of the invention, the siNA molecule is linked to alipophlic moiety. In anther embodiment, the lipophilic moiety ischolesterol. In another embodiment, the lipophilic moiety is attached to3′ end of the siNA molecule. In another embodiment, the lipophilicmoiety is attached to 5′ end of the siNA molecule. In anotherembodiment, a lipophilic moiety is attached to each of the 3′ and the 5′ends of the siNA molecule.

In some embodiments of the invention, all of the nucleotides of siNAmolecule portion of the myostatin siNA conjugates of the invention areunmodified. In other embodiments, the siNA molecules delivered by themethods of the present invention further comprise one or morenucleotides in either one or both strands of the molecule that arechemically-modified. Modifications include nucleic acid sugarmodifications, base modifications, backbone (internucleoside linkage)modifications, non-nucleotide modifications, and/or any combinationthereof. In certain instances, purine and pyrimidine nucleotides aredifferentially modified. For example, purine and pyrimidine nucleotidescan be differentially modified at the 2′-sugar position (i.e., at leastone purine has a different modification from at least one pyrimidine inthe same or different strand at the 2′-sugar position). In certaininstances the purines are unmodified in one or both strands, while thepyrimidines in one or both strands are modified. In certain otherinstances, the pyrimidines are unmodified in one or both strands, whilethe purines in one or both strands are modified. In some instances, atleast one modified nucleotide is a 2′-deoxy-2′-fluoro nucleotide, a2′-deoxy nucleotide, or a 2′-O-alkyl nucleotide. In some instances, atleast 5 or more of the pyrimidine nucleotides in one or both strands areeither all 2′-deoxy-2′-fluoro or all 2′-O-methyl pyrimidine nucleotides.In some instances, at least 5 or more of the purine nucleotides in oneor both strands are either all 2′-deoxy-2′-fluoro or all 2′-O-methylpurine nucleotides. In certain instances, wherein the siNA moleculescomprise one or more modifications as described herein, the nucleotidesat positions 1, 2, and 3 at the 5′ end of the guide (antisense) strandare unmodified. In certain embodiments, the siNA molecules delivered bythe methods of the present invention comprise one or more modifiedinternucleoside linking groups. In certain embodiments, eachinternucleoside linking group is, independently, a phosphodiester orphosphorothioate linking group.

In certain embodiments, the siNA molecule portion of the myostatin siNAconjugates of the invention have 3′ overhangs of one, two, three or fournucleotide(s) on one or both of the strands. In other embodiments, thedouble-stranded siNA molecules lack overhangs (i.e., have blunt ends).Preferably, the siNA molecule has 3′ overhangs of two nucleotides onboth the sense and antisense strands. The overhangs can be modified orunmodified. Examples of modified nucleotides in the overhangs include,but are not limited to, 2′-O-alkyl nucleotides, 2′-deoxy-2′-fluoronucleotides, locked nucleic acid (LNA) nucleotides, or 2′-deoxynucleotides. The overhanging nucleotides in the antisense strand cancomprise nucleotides that are complementary to nucleotides in themyostatin target sequence. Likewise, the overhangs in the sense strandcan comprise nucleotides that are present in the myostatin targetsequence. In certain instances, the siNA molecules have two 3′overhanging nucleotides on the antisense strand that are 2′-O-alkyl(e.g., 2′-O-methyl) nucleotides and two 3′ overhanging nucleotides onthe sense strand that are 2′-deoxy nucleotides. In other instances, thesiNA molecules have two 3′ overhanging nucleotides that are 2′-O-alkyl(e.g., 2′-O-methyl) nucleotides on both the antisense strand and thesense strand. In certain embodiments, the 2′-O-alkyl nucleotides are2′-O-methyl uridine nucleotides. In certain instances, the 3′ overhangsalso comprise one or more phosphorothioate linkages between nucleotidesof the overhang.

In some embodiments, the siNA molecule portion of the myostatin siNAconjugates of the invention have one or more terminal caps (alsoreferred to herein as “caps”). A cap may be present at the 3′-terminus(3′-cap) of the antisense strand (guide strand), at the 5′-terminus(5′-cap) of the sense strand (passenger strand), and/or at 3′-terminus(3′-cap) of the sense strand (passenger strand). The lipophilic moietymay be attached to the same terminus of the siNA molecule that containsa terminal cap.

In some embodiments, the siNA molecule portion of the myostatin siNAconjugates of the invention are phosphorylated at the 5′ end of theantisense strand. The phosphate group can be a phosphate, a diphosphateor a triphosphate.

In certain embodiments of this aspect of the invention, the siNA portionof the myotstatin siNA conjugates of the invention are double-strandedsiNA molecules wherein the antisense and/or sense strand comprises atleast one nucleotide sequence selected from SEQ ID NOs: 5-12, providedin Table 3. In a further embodiment, the siNA portion of the myostatinsiNA conjugates of the invention comprises any of the followingdouble-stranded molecules: SEQ ID NO: 5 and 6; SEQ ID NO: 7 and 8; SEQID NO: 9 and 10; or SEQ ID NO: 11 and 12.

The present invention further provides compositions comprising themyostatin siNA conjugates described herein with, optionally, apharmaceutically acceptable carrier or diluent. The methods of thepresent invention include delivery of compositions comprising themyostatin siNA conjugates described herein with a pharmaceuticallyacceptable carrier or diluent, wherein said compositions are formulatedfor systemic administration.

These and other aspects of the invention will be apparent upon referenceto the following Detailed Description and attached figures. Moreover, itis contemplated that any method or composition described herein can beimplemented with respect to any other method or composition describedherein and that different embodiments may be combined.

Additionally, patents, patent applications and other documents are citedthroughout the specification to describe and more specifically set forthvarious aspects of this invention. Each of these references cited hereinis hereby incorporated by reference in its entirety, including thedrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: In vivo screen of Mstn-cholesterol conjugates. FourMstn-cholesterol siRNAs (•), PBS (∘), and Placebo 5 non-targetingcontrol (•) were screened in CD-1 mice (n=5) at a 15 mpk dose by i.v.injection. (A) Mstn mRNA expression was determined based on ΔΔCtcalculations, relative to PBS, in gastrocnemius muscle 3 dayspost-injection. (B) Mstn protein levels were measured in serum 3 daysafter dosing. ***, P<0.001 (by one-way ANOVA).

FIG. 2: In vivo dose titration and duration of myostatin mRNA knockdown(KD) by Mstn-cholesterol siRNA. Mstn:1169-cholesterol (“Mstn-chol”)siRNA was tested in CD-1 mice (n=5) at 5, 15, and 50 mpk, in addition toPBS and Placebo 5 non-targeting controls (50 mpk) by i.v. injection.Mstn mRNA expression was determined based on ΔΔCt calculations, relativeto PBS, in gastrocnemius (A), tricep muscles (B), and EDL (C), at day 3,7, and 21 post-injection. (D) Serum Mstn protein levels were measured atindicated time points. *, P<0.05; **, P<0.01; ***, P<0.001 (by one-wayANOVA).

FIG. 3: Long-term myostatin knockdown (KD) leads to increase in musclesize. Mstn:1169-cholesterol siRNA (50 mpk) was injected intravenouslyinto CD-1 mice (n=12), in addition to PBS and Placebo 5 non-targetingcontrols. (A) Mstn mRNA levels, determined based on ΔΔCt calculations,relative to PBS, in gastrocnemius, EDL, quadriceps, triceps andspinotrapezius muscles at day 21 post-injection. (B) Serum Mstn proteinlevels, determined for the duration of the study. (C) Leg muscle size(maximum cross-sectional area), monitored for the duration of the studyand quantitated from a series of 10 micro-CT images using a customMATLAB and Definiens Developer XD software algorithm. (D) Gastrocnemiusmuscle weight of rested leg and exercised leg (used in situ musclefunction assay) at day 21. (E) Mean fiber cross-sectional area ofgastrocnemius muscle. (F) Mean total number of muscle fibers ingastrocnemius muscle. (G) Size frequency distribution of muscle fibersin gastrocnemius muscle. (H) Body weight measurements, determined forthe duration of the study. (I) Body composition analysis by qNMR(EchoMRI) at day 20. *, P<0.05; **, P<0.01; ***, P<0.001 ****, P<0.0001(by one-way ANOVA (a-b) or two-way ANOVA (c-h))

FIG. 4: Example of a muscle fatigue curve. Fatigue curves exhibit threestages of muscle fatigue: early fatigue, late fatigue and anon-fatigable stage. “Early fatigue” is represented by F_(max)-F₀,typically representative of type IIb fibers, which use creatinephosphate as an energy source. This stage is followed by “late fatigue”(F₀-F_(min)), typically representative of type IIa/x fibers, which useglycogen as an energy source. The final stage of the fatigue curve isthe “non-fatigable” stage (F_(min)), which is indicative of type Ifibers, which use fatty acids as an energy source.

FIG. 5: Long-term myostatin knockdown leads to changes in musclefunction. Mstn:1169 siRNA-cholesterol conjugate or Placebo50-cholesterol non-targeting siRNA control (50 mpk) were injectedintravenously into CD-1 mice (n=12). Muscle fatigue curves, force (A) orspecific force (B), were generated from in situ muscle function assayperformed on day 21/day 22 (n=9 total). Function parameters calculatedfrom the indicated fatigue curve are plotted below each curve in (A) and(B). Filled in bars indicate a statistical significant change in thespecified parameter between Mstn siRNA-cholesterol conjugate and Placebo5-cholesterol treatment. *, P<0.05; **, P<0.01; ***, P<0.001 ****,P<0.0001 (by one-way ANOVA (a-b) or two-way ANOVA (c-h))

FIG. 6: Heart weight measurements normalized to different parameters forPlacebo 5-chol and Mstn:1169 siRNA-conjugate. Mstn:1169 siRNA-conjugate(50 mpk) was injected intravenously into CD-1 mice (n=12), in additionto PBS and Placebo 5 cholesterol conjugate non-targeting control. Heartswere weighed and normalized to a variety of parameters (i.e., tibialength, body weight, lean mass, and muscle cross sectional area (CSA))after 21 days of Mstn knockdown to assess signs of cardiac hypertrophy.Filled in bars indicate a statistical significant change in thespecified parameter between Mstn siRNA-cholesterol conjugate and Placebo5-cholesterol treatment. *, P<0.05; ****, P<0.0001 (by one-way ANOVA(a-b) or two-way ANOVA (c-h))

FIG. 7: Ctnnb1-chol conjugates. (A) In vivo comparison of efficacy ofCtnnb1-chol conjugates in WT, LDLR−/−, and ApoE−/− mice. Ctnnb1-cholsiRNA was screened in CD-1 mice (n=3 or 4) at a 14 mpk dose by i.v.injection. Ctnnb1 mRNA expression was determined based on ΔΔCtcalculations, relative to PBS, in gastrocnemius muscle 3 dayspost-injection. **, P<0.01; ***, P<0.001 (by one-way ANOVA) (B)Ctnnb1-chol siRNAs with a single cholesterol attached at either the 3′or 5′ end of the passenger strand, were screened in CD-1 mice (n=5) at a15 mpk dose by i.v. injection. Ctnnb1 mRNA expression was compared toPBS and Placebo 5 non-targeting control (3′ chol). mRNA expression wasdetermined based on ΔΔCt calculations, relative to PBS, in gastrocnemiusmuscle 3 days post-injection. ***, P<0.001 (by one-way ANOVA).

DETAILED DESCRIPTION OF THE INVENTION A. Terms and Definitions

The following terminology and definitions apply as used in the presentapplication.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a cell”includes a combination of two or more cells, and the like.

Any concentration range, percentage range, ratio range or integer rangeis to be understood to include the value of any integer within therecited range, and when appropriate, fractions thereof (such as onetenth and one hundredth of an integer), unless otherwise indicated.

“About” or “approximately,” as used herein, in reference to a number aregenerally taken to include numbers that fall within a range of 5% ineither direction (greater than or less than) of the number unlessotherwise stated or otherwise evident from the context (except wheresuch number would exceed 100% of a possible value). Where ranges arestated, the endpoints are included within the range unless otherwisestated or otherwise evident from the context.

The phrases “2′-modified nucleotide,” “2′-substituted nucleotide” or anucleotide having a modification at the “2′-position” of the sugarmoiety, as used herein, generally refer to nucleotides comprising asubstituent at the 2′ carbon position of the sugar component that isother than H or OH. 2′-modified nucleotides include, but are not limitedto, bicyclic nucleotides wherein the bridge connecting two carbon atomsof the sugar ring connects the 2′ carbon and another carbon of the sugarring; and nucleotides with non-bridging 2′substituents, such as allyl,amino, azido, thio, O-allyl, OC₁₋₁₀ alkyl, —OCF3, O—(CH₂)₂—O—CH₃,2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)), orO—CH₂—C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is,independently, H or substituted or unsubstituted C₁₋₁₀ alkyl.2′-modified nucleotides may further comprise other modifications, forexample at other positions of the sugar and/or at the nucleobase. Thephrases “3′-modified nucleotide,” “3′-substituted nucleotide” or anucleotide having a modification at the “3′-position” of the sugarmoiety generally refers to a nucleotide comprising a modification,including a substituent, at the 3′ carbon position of the sugarcomponent.

The term “abasic” as used herein refers to its meaning as is generallyaccepted in the art. The term generally refers to sugar moieties lackinga nucleobase or having a hydrogen atom (H) or other non-nucleobasechemical groups in place of a nucleobase at the 1′ position of the sugarmoiety, see for example Adamic et al., U.S. Pat. No. 5,998,203. In oneembodiment, an siNA molecule of the invention may contain an abasicmoiety, wherein the abasic moiety is ribose, deoxyribose, ordideoxyribose sugar.

The term “acyclic nucleotide” as used herein refers to its meaning as isgenerally accepted in the art. The term generally refers to anynucleotide having an acyclic ribose sugar, for example where any of theribose carbon/carbon or carbon/oxygen bonds are independently or incombination absent from the nucleotide.

If no number of carbon atoms is specified, the term “alkyl” refers to asaturated aliphatic hydrocarbon group, branched or straight-chain,containing from 1 to 10 carbon atoms. An alkyl group can have a specificnumber of carbon atoms. For example, C₁-C₁₀, as in “C₁-C₁₀ alkyl” or“C₁₋₁₀ alkyl,” is defined to include groups having 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 carbons in a linear or branched arrangement. For example,“C₁-C₁₀ alkyl” specifically includes methyl, ethyl, n-propyl, i-propyl,n-butyl, t-butyl, i-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl,and so on. The term “cycloalkyl” means a monocyclic saturated aliphatichydrocarbon group having the specified number of carbon atoms. Forexample, “cycloalkyl” includes cyclopropyl, methyl-cyclopropyl,2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, cyclohexyl, and so on.

The term “antisense region” as used herein refers to its meaning as isgenerally accepted in the art. With reference to the siNA moleculesdelivered by the methods of the present invention, the term refers to anucleotide sequence of an siNA molecule having complementarity to amyostatin RNA. In addition, the antisense region of an siNA moleculecomprises a nucleic acid sequence having complementarity to a senseregion of the siNA molecule. In one embodiment, the antisense region ofan siNA molecule is referred to as the antisense strand or guide strand.

The term “biodegradable” as used herein refers to its meaning as isgenerally accepted in the art. The term generally refers to degradationin a biological system, for example, enzymatic degradation or chemicaldegradation.

The term “biological system” as used herein refers to its meaning as isgenerally accepted in the art. The term generally refers to material, ina purified or unpurified form, from biological sources including, butnot limited to, human or animal, wherein the system comprises thecomponents required for RNAi activity. Thus, the phrase includes, forexample, a cell, tissue, subject, or organism, or extract thereof. Theterm also includes reconstituted material from a biological source.

The term “blunt end” as used herein refers to its meaning as isgenerally accepted in the art. With reference to nucleic acid moleculesof the invention, the term refers to termini of a double-stranded siNAmolecule having no overhanging nucleotides. An siNA duplex molecule ofthe invention can comprise blunt ends at one or both termini of theduplex, such as termini located at the 5′-end of the antisense strand,the 5′-end of the sense strand, or both termini of the duplex.

The term “cap” (also referred to herein as “terminal cap”) as usedherein refers to its meaning as is generally accepted in the art. Withreference to exemplary nucleic acid molecules of the invention, the termrefers to a moiety, which can be a chemically-modified nucleotide or anon-nucleotide, incorporated at one or more termini of the nucleic acidmolecules of the invention. These terminal modifications may protect thenucleic acid molecule from exonuclease degradation and may help indelivery and/or localization of the nucleic acid molecule within a cell.The cap can be present at a 5′-terminus (5′-cap) or 3′-terminus (3′-cap)of a strand of the nucleic acid molecules of the invention, or can bepresent on both termini. For example, a cap can be present at the5′-end, 3′-end and/or 5′ and 3′-ends of the sense strand of a nucleicacid molecule of the invention. Additionally, a cap can be present atthe 3′-end of the antisense strand of a nucleic acid molecule of theinvention. In non-limiting examples, a 5′-cap includes, but is notlimited to, LNA; glyceryl; inverted deoxy abasic residue (moiety);4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide,4′-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitolnucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide;phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic3,5-dihydroxypentyl nucleotide; 3′-3′-inverted nucleotide moiety;3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety;3′-2′-inverted abasic moiety; 1,4-butanediol phosphate;3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate;3′-phosphorothioate; phosphorodithioate; or bridging or non-bridgingmethylphosphonate moiety. Non-limiting examples of a 3′-cap include, butare not limited to, LNA; glyceryl; inverted deoxy abasic residue(moiety); 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl)nucleotide; 4′-thio nucleotide; carbocyclic nucleotide; 5′-amino-alkylphosphate; 1,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate;6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropylphosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide;alpha-nucleotide; modified base nucleotide; phosphorodithioate;threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide;3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide,5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasic moiety;5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediol phosphate;5′-amino; bridging and/or non-bridging 5′-phosphoramidate;phosphorothioate and/or phosphorodithioate; bridging or non-bridgingmethylphosphonate; and 5′-mercapto moieties (for more details seeBeaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by referenceherein). In one embodiment, siNA molecules of the present inventioncontain a vinyl phosphate 5′ terminal cap, wherein carbon 5 of the sugarring contains the following substituent (═CH)—P(═O)(OH)_(2.)

The term “cell” as used herein refers to its meaning as is generallyaccepted in the art. The term is used herein in its usual biologicalsense, and does not refer to an entire multicellular organism, e.g.,specifically does not refer to a human being. The cell can be present inan organism, e.g., birds, plants and mammals, such as humans, cows,sheep, apes, monkeys, swine, dogs, and cats. The cell can be of somaticor germ line origin, totipotent or pluripotent, dividing ornon-dividing. The cell can also be derived from or can comprise a gameteor embryo, a stem cell, or a fully differentiated cell. The cell can bea muscle cell.

The phrases “chemically-modified nucleotide,” “modified nucleotide” or,when used in reference to nucleotides within the myostatin siNAmolecules described herein, “chemical modification,” refer to anucleotide that contains a modification in the chemical structure of theheterocyclic base moiety, sugar and/or phosphate of the unmodified (ornatural) nucleotide as is generally known in the art (i.e., at least onemodification compared to a naturally occurring RNA or DNA nucleotide).In certain embodiments, the terms can refer to certain forms of RNA thatare naturally occurring in certain biological systems, for example2′-O-methyl modifications or inosine modifications. A modifiednucleotide includes abasic nucleotides. Modified nucleotides includenucleotides with a modified sugar ring or sugar surrogate. Modifiedheterocyclic base moieties include without limitation, universal bases,hydrophobic bases, promiscuous bases, size-expanded bases, andfluorinated bases as defined herein. Certain of these nucleobases areparticularly useful for increasing the binding affinity of the siNAmolecules as provided herein. These include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. Amodified internucleoside linkage refers to any internucleoside linkageother than a naturally occurring internucleoside linkage. Non-limitingexamples of modified nucleotides are described herein and in U.S.application Ser. No. 12/064,014 (published as US 20090176725).

The terms “complementarity” or “complementary” as used herein refers toits meaning as is generally accepted in the art. The terms generallyrefer to the formation or existence of hydrogen bond(s) between onenucleic acid sequence and another nucleic acid sequence by eithertraditional Watson-Crick or other non-traditional types of bonding asdescribed herein. In reference to the nucleic molecules delivered by themethods of the present invention, the binding free energy for a nucleicacid molecule with its complementary sequence is sufficient to allow therelevant function of the nucleic acid to proceed, e.g., RNAi activity.Determination of binding free energies for nucleic acid molecules iswell known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant.Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785).Perfect complementary means that all the contiguous residues of anucleic acid sequence will hydrogen bond with the same number ofcontiguous residues in a second nucleic acid sequence. Partialcomplementarity can include various mismatches or non-based pairednucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mismatches,non-nucleotide linkers, or non-based paired nucleotides) within thenucleic acid molecule, which can result in bulges, loops, or overhangsbetween the sense strand or sense region and the antisense strand orantisense region of a nucleic acid molecule or between the antisensestrand or antisense region of a nucleic acid molecule and acorresponding target nucleic acid molecule. Such partial complementaritycan be represented by a % complementarity that is determined by thenumber of non-base paired nucleotides, e.g., about 50%, 60%, 70%, 80%,90% etc. depending on the total number of nucleotides involved. Suchpartial complementarity is permitted to the extent that the nucleic acidmolecule (e.g., siNA) maintains its function, for example the ability tomediate sequence specific RNAi.

The terms “composition” or “formulation” as used herein refer to theirgenerally accepted meaning in the art. These terms generally refer to acomposition or formulation, such as in a pharmaceutically acceptablecarrier or diluent, in a form suitable for administration, e.g.,systemic administration, into a cell or subject, including, for example,a human. Suitable forms, in part, depend upon the use or the route ofentry, for example oral, transdermal, inhalation, or by injection. Suchforms should not prevent the composition or formulation from reaching atarget cell (i.e., a cell to which the negatively charged nucleic acidis desirable for delivery). For example, compositions injected into theblood stream should be soluble. Other factors are known in the art, andinclude considerations such as toxicity and forms that prevent thecomposition or formulation from exerting its effect. As used herein,pharmaceutical formulations include formulations for human andveterinary use. A “pharmaceutically acceptable composition” or“pharmaceutically acceptable formulation” can refer to a composition orformulation that allows for the effective distribution of the nucleicacid molecules of the instant invention to the physical location mostsuitable for their desired activity.

The term “conjugate” refers to an atom or group of atoms bound to ansiNA molecule delivered by the methods of the invention. In general,conjugate groups modify one or more properties of the molecule to whichthey are attached, including, but not limited to pharmacodynamics,pharmacokinetic, binding, absorption, cellular distribution, cellularuptake, charge and clearance. Conjugate groups are routinely used in thechemical arts and are linked directly or via an optional linking moietyor linking group to the parent compound, such as an siNA molecule. ThesiNA conjugates used in the methods of the present invention comprise ansiNA molecule that targets myostatin RNA linked to a lipophilic moiety,such as cholesterol. In certain embodiments, the lipophilic moiety isattached to a 3′ or 5′ terminal nucleotide or to an internal nucleotideof a myostatin siNA molecule. As used herein, “conjugate linking group”refers to any atom or group of atoms used to attach the lipophilicmoiety to a myostatin siNA molecule. Linking groups or bifunctionallinking moieties such as those known in the art are amenable to thepresent invention.

The terms “detecting” or “measuring,” as used herein in connection withan activity, response or effect, indicate that a test for detecting ormeasuring such activity, response, or effect is performed. Suchdetection and/or measuring may include values of zero. Thus, if a testfor detection or measuring results in a finding of no activity (activityof zero), the step of detecting or measuring the activity hasnevertheless been performed.

The phrase “effective amount” as used herein refers to its meaning as isgenerally accepted in the art. The term generally refers to the amountof a molecule, compound or composition that will elicit the intendedbiological response of a cell, tissue, system, animal or human that isbe sought by the researcher, veterinarian, medical doctor or otherclinician (e.g., reduction in myostatin protein levels, as measured inmuscle tissue or serum). A “therapeutically effective amount” generallyrefers to the amount of a molecule, compound or composition that willelicit the medical response if a given clinical treatment is consideredeffective when there is at least a 25% reduction in a measurableparameter associated with a disease or disorder, a therapeuticallyeffective amount of a drug for the treatment of that disease or disorderis that amount necessary to effect at least a 25% reduction in thatparameter.

The term “expression” as used herein refers to its meaning as isgenerally accepted in the art. The term generally refers to the processby which a gene ultimately results in a protein. Expression includes,but is not limited to, transcription, splicing, post-transcriptionalmodification and translation.

The term “gene” as used herein refers to its meaning as is generallyaccepted in the art. The term generally refers to a nucleic acid (e.g.,DNA or RNA) sequence that comprises partial length or entire lengthcoding sequences necessary for the production of a polypeptide. A genecan also include the UTR or non-coding region of the nucleic acidsequence. A gene can also encode a functional RNA (fRNA) or non-codingRNA (ncRNA), such as small temporal RNA (stRNA), micro RNA (miRNA),small nuclear RNA (snRNA), short interfering RNA (siRNA), smallnucleolar RNA (snRNA), ribosomal RNA (rRNA), transfer RNA (tRNA) andprecursor RNAs thereof. Such non-coding RNAs can serve as target nucleicacid molecules for siNA mediated RNA interference in modulating theactivity of fRNA or ncRNA involved in functional or regulatory cellularprocesses. Aberrant fRNA or ncRNA activity leading to disease cantherefore be modulated by delivery of myostatin-directed siNA moleculesby the methods of the invention. siNA molecules targeting fRNA and ncRNAcan also be used to manipulate or alter the genotype or phenotype of asubject, organism or cell, by intervening in cellular processes such asgenetic imprinting, transcription, translation, or nucleic acidprocessing (e.g., transamination, methylation etc.). The term “gene” canbe used when referencing a gene to which an siNA molecule, delivered bythe methods of present invention, is either directly (i.e., the siNAmolecule comprises an antisense strand having partial or completecomplementarity to the gene) or indirectly (i.e., the siNA moleculecomprises an antisense strand having partial or complete complementarityto a gene in the expression or activity pathway of the gene) targeted.

The term “heteroaryl,” as used herein, represents a stable monocyclic orbicyclic ring of up to 7 atoms in each ring, wherein at least one ringis aromatic and contains from 1 to 4 heteroatoms selected from the groupconsisting of O, N and S. Heteroaryl groups within the scope of thisdefinition include but are not limited to: acridinyl, carbazolyl,cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, furanyl,thienyl, benzothienyl, benzofuranyl, benzimidazolonyl, benzoxazolonyl,quinolinyl, isoquinolinyl, dihydroisoindolonyl, imidazopyridinyl,isoindolonyl, indazolyl, oxazolyl, oxadiazolyl, isoxazolyl, indolyl,pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl,tetrahydroquinoline. “Heteroaryl” is also understood to include theN-oxide derivative of any nitrogen-containing heteroaryl. In cases wherethe heteroaryl substituent is bicyclic and one ring is non-aromatic orcontains no heteroatoms, it is understood that attachment is via thearomatic ring or via the heteroatom containing ring, respectively.

The term “heterocycle,” as used herein, is intended to mean a 3- to10-membered aromatic or nonaromatic heterocycle containing from 1 to 4heteroatoms selected from the group consisting of O, N and S, andincludes bicyclic groups. “Heterocycle” includes the above mentionedheteroaryls, as well as dihydro and tetrahydro analogs thereof. Furtherexamples of “heterocycle” include, but are not limited to the following:azetidinyl, benzoimidazolyl, benzofuranyl, benzofurazanyl,benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl,carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl,indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl,isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl,oxooxazolidinyl, oxazolyl, oxazoline, oxopiperazinyl, oxopyrrolidinyl,oxomorpholinyl, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl,pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl,quinazolinyl, quinolyl, quinoxalinyl, tetrahydropyranyl,tetrahydrofuranyl, tetrahydrothiopyranyl, tetrahydroisoquinolinyl,tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl,triazolyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl,pyridin-2-onyl, pyrrolidinyl, morpholinyl, thiomorpholinyl,dihydrobenzoimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl,dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl,dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl,dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,dioxidothiomorpholinyl, methylenedioxybenzoyl, tetrahydrofuranyl, andtetrahydrothienyl, and N-oxides thereof. Attachment of a heterocyclylsubstituent can occur via a carbon atom or via a heteroatom.

The terms “including” (and any form thereof, such as “includes” and“include”), “comprising” (and any form thereof, such as “has” or “have”)or “containing” (and any form thereof (“contains” or “contain”) areinclusive and open-ended and do not exclude additional, unrecitedelements or method steps.

The terms “inhibit,” “down-regulate,” or “reduce” as used herein referto their meanings as generally accepted in the art. With reference tothe siNA molecules delivered by the methods of the present invention,the term generally refers to reduction in the expression of a gene, orin the level of RNA molecules encoding one or more proteins or proteinsubunits, or in the activity of one or more proteins or proteinsubunits, below that observed in the absence of the siNA molecules.Down-regulation can be associated with post-transcriptional silencing,such as RNAi mediated cleavage.

The terms “intermittent” or “intermittently” as used herein refer totheir meaning as generally accepted in the art. The terms generallyrefer to periodic stopping and starting at either regular or irregularintervals.

The terms “internucleoside linkage,” “internucleoside linker,”“internucleoside linking group,” “internucleotide linkage,”“internucleotide linker” or “internucleotide linking group” are usedherein interchangeably and refer to any linker or linkage between twonucleoside (i.e., a heterocyclic base moiety and a sugar moiety) units,as is known in the art, including, for example, but not as limitation,phosphate, analogs of phosphate, phosphonate, guanidium, hydroxylamine,hydroxylhydrazinyl, amide, carbamate, alkyl, and substituted alkyllinkages. Internucleoside linkages constitute the backbone of a nucleicacid molecule. In one aspect, a nucleotide of an siNA molecule may belinked to a consecutive nucleotide through a linkage between the3′-carbon of the sugar of the first nucleotide and the sugar moiety ofthe second nucleotide (herein referred to as a 3′ internucleosidelinkage). A 3′-5′ internucleoside linkage, as used herein, refers to aninternucleoside linkage that links two consecutive nucleoside units,wherein the linkage is between the 3′ carbon of the sugar moiety of thefirst nucleoside and the 5′ carbon of the sugar moiety of the secondnucleoside. In another aspect, a nucleotide of an siNA molecule may belinked to a consecutive nucleotide through a linkage between the2′-carbon of the sugar of the first nucleotide and the sugar moiety ofthe second nucleotide (herein referred to as a 2′ internucleosidelinkage). A 2′-5′ internucleoside linkage, as used herein, refers to aninternucleoside linkage that links two consecutive nucleoside units,wherein the linkage is between the 2′ carbon of the sugar moiety of thefirst nucleoside and the 5′ carbon of the sugar moiety of the secondnucleoside.

The term “linker” or “spacer,” as used herein, refers to their meaningas generally accepted in the art. Generally, they refer to any moleculethat links or joins components. In the case of the present invention, alinker or spacer may be used to join a myostatin siNA molecule to alipophilic molecule to form a myostatin siNA conjugate. The linker canbe a nucleic acid or a non-nucleic acid-based linker. The term“biodegradable linker” refers to an optional linker molecule designed toconnect the siNA molecule to the lipophilic moiety and which issusceptible to degradation in a biological system.

The term “livestock,” in reference to animals, refers to domesticatedanimals, semi-domesticated animals or captive wild animals that areraised in an agricultural setting to produce commodities such as food,fiber and labor. Livestock animals include cattle, swine, fowl (e.g.,chicken, turkey), sheep, bison, goats and the like.

The phrase “metered dose inhaler” or “MDI” refers to a unit comprising acan, a secured cap covering the can, and a formulation metering valvesituated in the cap. MDI systems include a suitable channeling device.Suitable channeling devices comprise for example, a valve actuator and acylindrical or cone-like passage through which medicament can bedelivered from the filled canister via the metering valve to the nose ormouth of a patient such as a mouthpiece actuator.

The term “microRNA” or “miRNA” as used herein refers to its meaning asis generally accepted in the art. The term generally refers to a smallnon-coding RNA that regulates the expression of target messenger RNAseither by mRNA cleavage, translational repression/inhibition orheterochromatic silencing (see for example Ambros, 2004, Nature, 431,350-355; Bartel, 2004, Cell, 116, 281-297; Cullen, 2004, Virus Research,102, 3-9; He et al., 2004, Nat. Rev. Genet., 5, 522-531; Ying et al.,2004, Gene, 342, 25-28; and Sethupathy et al., 2006, RNA, 12:192-197).The phenomenon of RNA interference includes the endogenously inducedgene silencing effects of miRNAs. As used herein, “microRNA mimetic”refers to an siNA molecule having a sequence that is at least partiallyidentical to that of a microRNA. In certain embodiments, a microRNAmimetic comprises the microRNA seed region of a microRNA. In certainembodiments, a microRNA mimetic modulates translation of more than onetarget nucleic acid.

The term “modulate” or “modulation” as used herein refers to its meaningas is generally accepted in the art. With reference to nucleic acidmolecules delivered by the methods of the present invention, the termrefers to when the expression of a gene, or the level of one or more RNAmolecules (coding or non-coding), or the activity of one or more RNAmolecules or proteins or protein subunits, is up-regulated ordown-regulated, such that expression level or activity is greater thanor less than that observed in the absence of the molecule that effectsmodulation. For example, the term “modulate” in some embodiments canrefer to inhibition and, in other embodiments, can refer to potentiationor up-regulation, e.g., of gene expression.

The phrases “muscle cell” or “muscle tissue” as used herein refers totheir meaning as is generally accepted in the art. They refer to a cellor group of cells derived from muscle of any kind (for example, skeletalmuscle and smooth muscle, e.g. from the digestive tract, urinarybladder, blood vessels or cardiac tissue). Such muscle cells may bedifferentiated or undifferentiated, such as myoblasts, myocytes,myotubes, cardiomyocytes and cardiomyoblasts.

The phrase “myostatin siNA molecule” or “myostatin siNA” as used hereinrefers to a siNA molecule that targets a myostatin gene. The phrase“myostatin siNA conjugate” as used herein refers to a siNA molecule thattargets a myostatin gene and is linked to a lipophilic moiety.

The phrase “non-base paired” refers to nucleotides that are not basepaired between the sense strand or sense region and the antisense strandor antisense region of a double-stranded siNA molecule. Non-base pairednucleotides can include, for example, but not as limitation, mismatches,overhangs, and single stranded loops.

The term “non-nucleotide” refers to any group or compound that can beincorporated into a polynucleotide chain in the place of one or morenucleotide units, such as for example but not limitation, abasicmoieties or alkyl chains. The group or compound is “abasic” in that itdoes not contain a commonly recognized nucleotide base, such asadenosine, guanine, cytosine, uracil or thymine and, therefore, lacks anucleobase at the 1′-position.

The term “nucleobase” is used herein to refer to the heterocyclic baseportion of a nucleotide. Nucleobases may be naturally occurring or maybe modified. In certain embodiments, a nucleobase may comprise any atomor group of atoms capable of hydrogen bonding to a base of anothernucleic acid.

The term “nucleotide” is used as is generally recognized in the art.Nucleotides generally comprise a heterocyclic base moiety (i.e., anucleobase), a sugar, and an internucleoside linkage, e.g., a phosphate.The base can be a natural base (standard), a modified base, or a baseanalog, as are well known in the art. Such bases are generally locatedat the 1′ position of a nucleotide sugar moiety. Additionally, thenucleotides can be unmodified or modified at the sugar, internucleosidelinkage, and/or base moiety, (also referred to interchangeably asnucleotide analogs, modified nucleotides, non-natural nucleotides,non-standard nucleotides and others; see, for example, U.S. applicationSer. No. 12/064,014 (published as US 20090176725)). A naturallyoccurring internucleoside linkage refers to a 3′ to 5′ phosphodiesterlinkage (also referred to herein as a 3′-5′ phosphodiester linkage).

The term “overhang” as used herein refers to its meaning as is generallyaccepted in the art. With reference to exemplary double-stranded nucleicacid molecules delivered by the methods of the present invention, theterm generally refers to the terminal portion of a nucleotide sequencethat is not base-paired between the two strands of a double-strandednucleic acid molecule. Overhangs, when present, are typically at the3′-end of one or both strands in an siNA duplex.

The phrase “pharmaceutically acceptable carrier or diluent” as usedherein refers to its meaning as it generally accepted in the art. Thephrase generally refers to any substance suitable for use inadministering to a subject, such as an animal. In certain embodiments, apharmaceutically acceptable carrier or diluent is sterile saline. Incertain embodiments, such sterile saline is pharmaceutical grade saline.

The term “phosphorothioate” refers to an internucleoside phosphatelinkage comprising one or more sulfur atoms in place of an oxygen atom.Hence, the term phosphorothioate refers to both phosphorothioate andphosphorodithioate internucleoside linkages.

The term “ribonucleotide” as used herein refers to its meaning as isgenerally accepted in the art. The term generally refers to a nucleotidewith a hydroxyl group at the 2′ position of a β-D-ribofuranose moiety.

The term “RNA” as used herein refers to its generally accepted meaningin the art. Generally, the term RNA refers to a molecule comprising atleast one ribofuranoside moiety. The term can include double-strandedRNA, single-stranded RNA, isolated RNA such as partially purified RNA,essentially pure RNA, synthetic RNA, recombinantly produced RNA, as wellas altered RNA that differs from naturally occurring RNA by theaddition, deletion, substitution and/or alteration of one or morenucleotides. Such alterations can include addition of non-nucleotidematerial, such as to the end(s) of an siNA molecule or internally, forexample at one or more nucleotides of the RNA. Nucleotides in thenucleic acid molecules of the instant invention can also comprisenon-standard nucleotides, such as non-naturally occurring nucleotides orchemically synthesized nucleotides or deoxynucleotides. These alteredRNAs can be referred to as analogs or analogs of naturally-occurringRNA.

The phrase “RNA interference” or term “RNAi” refer to the biologicalprocess generally known in the art of inhibiting or down regulating geneexpression in a cell, typically by causing destruction of specifictarget RNA and mediated by sequence-specific nucleic acid molecules(e.g., short interfering nucleic acid molecule), see for example Zamoreand Haley, 2005, Science, 309, 1519-1524; Vaughn and Martienssen, 2005,Science, 309, 1525-1526; Zamore et al., 2000, Cell, 101, 25-33; Bass,2001, Nature, 411, 428-429; Elbashir et al., 2001, Nature, 411, 494-498;and Kreutzer et al., International PCT Publication No. WO 00/44895;Zernicka-Goetz et al., International PCT Publication No. WO 01/36646;Fire, International PCT Publication No. WO 99/32619; Plaetinck et al.,International PCT Publication No. WO 00/01846; Mello and Fire,International PCT Publication No. WO 01/29058; Deschamps-Depaillette,International PCT Publication No. WO 99/07409; and Li et al.,International PCT Publication No. WO 00/44914; Allshire, 2002, Science,297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein,2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297,2232-2237; Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus etal., 2002, RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16,1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831).Additionally, the term RNAi is meant to be equivalent to other termsused to describe sequence specific RNA interference, such as posttranscriptional gene silencing, translational inhibition,transcriptional inhibition, or epigenetics. For example, siNA moleculesof the invention can be used to epigenetically silence genes at eitherthe post-transcriptional level or the pre-transcriptional level. In anon-limiting example, epigenetic modulation of gene expression by siNAmolecules delivered by the methods of the present invention can resultfrom siNA mediated modification of chromatin structure or methylationpatterns to alter gene expression (see, for example, Verdel et al.,2004, Science, 303, 672-676; Pal-Bhadra et al., 2004, Science, 303,669-672; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002,Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; andHall et al., 2002, Science, 297, 2232-2237). Modulation of geneexpression by siNA molecules delivered by the methods of the presentinvention can result from siNA mediated cleavage of RNA (either codingor non-coding RNA) via RISC.

The phrase “sense region” as used herein refers to its meaning as isgenerally accepted in the art. With reference to siNA moleculesdescribed herein, the term refers to a nucleotide sequence of an siNAmolecule having complementarity to an antisense region of the siNAmolecule. In addition, the sense region of a siNA molecule can comprisea nucleic acid sequence having homology or sequence identity with atarget nucleic acid sequence. In one embodiment, the sense region of thesiNA molecule is also referred to as the sense strand or passengerstrand.

The phrases “short interfering nucleic acid,” “siNA,” “siNA molecule,”“short interfering RNA,” “siRNA,” “short interfering nucleic acidmolecule,” “short interfering oligonucleotide molecule,” or “chemicallymodified short interfering nucleic acid molecule” refer to any nucleicacid molecule capable of inhibiting or down regulating gene expressionor viral replication by mediating RNA interference (“RNAi”) in asequence-specific manner. These terms can refer to both individualnucleic acid molecules, a plurality of such nucleic acid molecules, orpools of such nucleic acid molecules. The siNA can be a symmetric orasymmetric double-stranded nucleic acid molecule comprisingself-complementary sense and antisense strands or regions, wherein theantisense strand/region comprises a nucleotide sequence that iscomplementary to a nucleotide sequence in a target nucleic acid moleculeor a portion thereof, and the sense strand/region comprises a nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof. A symmetric duplex refers to an siNA molecule comprising senseand antisense regions each comprising the same number of nucleotides. Anasymmetric duplex refers to an siNA molecule comprising an antisenseregion and a sense region that comprises fewer nucleotides than theantisense region, to the extent that the sense region has enoughcomplementary nucleotides to base pair with the antisense region to forma duplex. For example, an asymmetric double-stranded siNA molecule cancomprise an antisense region having length sufficient to mediate RNAi ina cell or in vitro system, e.g. about 15 to about 30, and a sense regionhaving about 3 to about 25 nucleotides that are complementary to theantisense region. As an example, an asymmetric double-stranded hairpinsiNA molecule can also comprise a loop region comprising about 4 toabout 12 nucleotides. The loop portion of an asymmetric hairpin siNAmolecule can comprise nucleotides, non-nucleotides, linker molecules, orconjugate molecules as described herein. An siNA molecule can alsocomprise a single-stranded polynucleotide having a nucleotide sequencecomplementary to a portion of a nucleotide sequence in a target nucleicacid molecule (for example, where such siNA molecule does not requirethe presence within the siNA molecule of a nucleotide sequencecorresponding to the target nucleic acid sequence or a portion thereof).A single-stranded siNA molecule is an RNAi molecule, functioning throughan RNAi mechanism.

The term “subject” as used herein refers to its meaning as is generallyaccepted in the art. As used herein, term generally refers to anorganism to which the siNA conjugates as described and compositionsthereof can be administered. The term “subject” is intended to includehuman and non-human animals. Non-human animals include all vertebrates,e.g. mammals and non-mammals, such as non-human primates, sheep, dogs,cats, cows, horses, chickens, amphibians, rabbits, hamsters, guineapigs, livestock and reptiles. A subject can be an organism that has beenpreviously identified as a suitable candidate for administration of thesiNA conjugates as per the methods of the invention. For example, asubject can be a mammal, such as a human, diagnosed with amusculoskeletal disease, wherein it is believed that treatment with thesiNA conjugates described herein has potential of resulting in apositive clinical outcome. The term “subject” is also intended toinclude an embryo, including a chicken embryo contained within an egg.

The term “sugar moiety” means a natural or modified sugar ring or sugarsurrogate.

The term “sugar surrogate” generally refers to a structure that iscapable of replacing the furanose ring of a naturally occurringnucleotide. In certain embodiments, sugar surrogates are non-furanose(or 4′-substituted furanose) rings or ring systems or open systems. Suchstructures include simple changes relative to the natural furanose ring,such as a 6-membered ring or may be more complicated as is the case withthe non-ring system used in peptide nucleic acid. Sugar surrogatesincludes without limitation morpholinos, cyclohexenyls andcyclohexitols. In most nucleotides having a sugar surrogate group, theheterocyclic base moiety is generally maintained to permithybridization.

The phrase “systemic administration” as used herein refers to itsmeaning as is generally accepted in the art. The term generally refersto methods or techniques of administering a molecule, drug, agent orcompound in a manner resulting in in vivo systemic absorption oraccumulation of drugs in the blood stream followed by distributionthroughout the entire body. Systemic administration includes in ovoadministration.

The term “target” cellular protein, peptide, or polypeptide, orpolynucleotide or nucleic acid (such as “target DNA,” “target RNA,”“target nucleic acid”), as used herein, refers to a protein or nucleicacid, respectively, of which an siNA molecule may be capable ofinhibiting or down regulating the expression. In certain embodiments,target RNA is mRNA, pre-mRNA, non-coding RNA, pri-microRNA,pre-microRNA, mature microRNA, promoter-directed RNA, or naturalantisense transcripts. As used herein, “target mRNA” refers to apre-selected RNA molecule that encodes a protein. As used herein,“target pre-mRNA” refers to a pre-selected RNA transcript that has notbeen fully processed into mRNA. Notably, pre-RNA includes one or moreintron. As used herein, “target microRNA” refers to a pre-selectednon-coding RNA molecule about 18-30 nucleobases in length that modulatesexpression of one or more proteins or to a precursor of such anon-coding molecule. As used herein, “target non-coding RNA” refers to apre-selected RNA molecule that is not translated to generate a protein.Certain non-coding RNA is involved in regulation of expression.

The phrases “target site,” “target sequence” and “target nucleic acidsite” as used herein refer to their meanings as generally accepted inthe art. The term generally refers to a sequence within a target nucleicacid (e.g., RNA) that is “targeted,” e.g., for cleavage mediated by ansiNA molecule that contains sequences within its antisense region thatare complementary to the target sequence.

The phrase “universal base” as used herein refers to its meaning as isgenerally accepted in the art. The term universal base generally refersto nucleotide base analogs that form base pairs with each of the naturalDNA/RNA bases with little or no discrimination between them.Non-limiting examples of universal bases include C-phenyl, C-naphthyland other aromatic derivatives, inosine, azole carboxamides, andnitroazole derivatives such as 3-nitropyrrole, 4-nitroindole,5-nitroindole, and 6-nitroindole as known in the art (see, for example,Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

The term “up-regulate” as used herein refers to its meaning as isgenerally accepted in the art. With reference nucleic acid moleculesdescribed herein, the term refers to an increase in either theexpression of a gene, or the level of RNA molecules or equivalent RNAmolecules encoding one or more proteins or protein subunits, or theactivity of one or more RNAs, proteins or protein subunits, above thatobserved in the absence of the nucleic acid molecules (e.g., siNA) ofthe invention. In certain instances, up-regulation or promotion of geneexpression with an siNA molecule is above that level observed in thepresence of an inactive or attenuated molecule. In other instances,up-regulation or promotion of gene expression with siNA molecules isabove that level observed in the presence of, for example, an siNAmolecule with scrambled sequence or with mismatches. In still otherinstances, up-regulation or promotion of gene expression with a nucleicacid molecule of the instant invention is greater in the presence of thenucleic acid molecule than in its absence. In some instances,up-regulation or promotion of gene expression is associated withinhibition of RNA mediated gene silencing, such as RNAi mediatedcleavage or silencing of a coding or non-coding RNA target thatdown-regulates, inhibits, or silences the expression of the gene ofinterest to be up-regulated. The down-regulation of gene expression can,for example, be induced by a coding RNA or its encoded protein, such asthrough negative feedback or antagonistic effects. The down-regulationof gene expression can, for example, be induced by a non-coding RNAhaving regulatory control over a gene of interest, for example bysilencing expression of the gene via translational inhibition, chromatinstructure, methylation, RISC mediated RNA cleavage, or translationalinhibition. As such, inhibition or down-regulation of targets thatdown-regulate, suppress, or silence a gene of interest can be used toup-regulate expression of the gene of interest toward therapeutic use.

The term “vector” as used herein refers to its meaning as is generallyaccepted in the art. The term vector generally refers to any nucleicacid- and/or viral-based expression system or technique used to deliverone or more nucleic acid molecules.

B. Myostatin siNA Molecules

Myostatin is a known growth factor involved in regulation of musclegrowth. In particular, myostatin is a member of the TGF-β family ofgrowth factors and is a potent negative regulator of myogenesis.Knock-out mice for myostatin have greatly increased muscle mass overtheir entire body, having approximately 30% greater body weight thannormal mice, and exhibiting a 2 to 3 fold increase in individual muscleweights due to muscle fiber hyperplasia and hypertrophy. Naturalmutations in myostatin have been identified as being responsible for the“double-muscled” phenotype, such as the Belgian Blue and Piedmontesecattle breeds. See McPherron, A. C. et al, 1997, Nature 387:83-92;McPherron, A. C. et al., 1997, Proc. Natl. Acad. Sci. USA94:12457-12461; Kambadur, R. et al., 1997, Genome Res. 7:910-916;Grobet, L. et al., 1997, Nat. Genet. 17:71-74).

The siNA molecules delivered by the methods of the present invention aredesigned to target a myostatin gene. The siNA molecules may be directedto a myostatin gene sequence derived from an array of suitable animals,including for example human, cattle, pigs, fowl or mouse. For example, amyostatin siNA molecule delivered by the methods of the presentinvention may be designed to target a myostatin mRNA as set forth inTable 1:

TABLE 1 NCBI GenBank Species Accession No. Bos taurus GQ184147 Bosindicius AY794986 Home sapien AF104922 Sus scrofa AY448008 Equuscaballus AB033541 Gallus gallus AY448007 Meleagris gallopavo AF019625Ovis aries AM992883 Capra hircus GQ246167 Macaca fascicularis AY055750Mus musculus NM_010834

The instant invention features single- or double-stranded siNA moleculesthat target a myostatin gene, lipophilic conjugates thereof, and methodsof delivering and using the same in vivo, wherein said delivered siNAmolecules are capable of mediating RNA interference. The antisensestrand (or guide strand) of the siNA portion of a myostatin siNAconjugate is complementary to a myostatin target nucleic acid. The siNAmolecule portion of the conjugates can take different oligonucleotideforms, including but not limited to short interfering RNA (siRNA),double-stranded RNA (dsRNA), micro-RNA (miRNA) and short hairpin RNA(shRNA) molecules. In certain embodiments, the siNA molecule issingle-stranded. In other embodiments, the siNA molecule isdouble-stranded molecules, wherein said double-stranded moleculecomprises an antisense strand and a sense strand. The myostatin siNAmolecules comprised within the myostatin siNA conjugates modulateexpression of a myostatin target nucleic acid. In one embodiment, thesiNA molecules inhibit or reduce expression of a myostatin targetnucleic acid. In one aspect, the siNA molecule portion of the myostatinsiNA conjugates is single-stranded, wherein the single oligonucleotidestrand comprises a sequence that is complementary to at least a part ofa myostatin nucleic acid associated with myostatin gene expression. Forpurposes of this disclosure, the single strand of a single-stranded siNAmolecule is referred to as the antisense strand.

In another aspect, the siNA molecule portion of the myostatin siNAconjugates is a double-stranded siNA molecule, wherein a double-strandedsiNA molecule comprises a sense and an antisense oligonucleotide strand.The antisense strand comprises a sequence that is complementary to atleast a part of a myostatin target nucleic acid associated withmyostatin gene expression, and the sense strand is complementary to theantisense strand. The double-stranded siNA molecules can comprise twodistinct and separate strands that can be symmetric or asymmetric andare complementary, i.e., two single-stranded oligonucleotides, or cancomprise one single-stranded oligonucleotide in which two complementaryportions, e.g., a sense region and an antisense region (which, in thiscontext, will be referred to herein as a sense strand and an antisensestrand, respectively), are base-paired, and are covalently linked by oneor more single-stranded “hairpin” areas (i.e. loops) resulting in, forexample, a short-hairpin polynucleotide. The linker can be apolynucleotide linker or a non-nucleotide linker. In some embodiments,the linker is a non-nucleotide linker. In some embodiments, a hairpinsiNA molecule contains one or more loop motifs, wherein at least one ofthe loop portions of the siNA molecule is biodegradable. For example, ashort hairpin siNA molecule can be designed such that degradation of theloop portion of the siNA molecule in vivo can generate a double-strandedsiNA molecule with 3′-terminal overhangs, such as 3′-terminal nucleotideoverhangs comprising 1, 2, 3 or 4 nucleotides.

The antisense strand of the siNA molecule portion of the describedconjugates is complementary to a portion of a myostatin target nucleicacid sequence. In some embodiments, the target nucleic acid is selectedfrom a myostatin target mRNA, a myostatin target pre-mRNA, a myostatintarget microRNA, and a myostatin target non-coding RNA. In certainembodiments, the antisense strand of the siNA molecule comprises aregion that is 100% complementarity to a myostatin target nucleic acidsequence and wherein the region of 100% complementarity is at least 10nucleobases. In certain embodiments, the region of 100% complementarityis at least 15 nucleobases. In certain embodiments, the region of 100%complementarity is at least 20 nucleobases. In certain embodiments, theregion of 100% complementarity is at least 25 nucleobases. In certainembodiments, the region of 100% complementarity is at least 30nucleobases. In certain embodiments, the antisense strand of the siNAmolecule is at least 85% complementary to a myostatin target nucleicacid sequence. In certain embodiments, the antisense strand is at least90% complementary to a myostatin target nucleic acid sequence. Incertain embodiments, the antisense strand is at least 95% complementaryto a myostatin target nucleic acid sequence. In certain embodiments, theantisense strand is at least 98% complementary to a myostatin targetnucleic acid sequence. In certain embodiments, the antisense strand is100% complementary to a myostatin target nucleic acid sequence. Thecomplementary nucleotides may or may not be contiguous nucleotides. Inone embodiment, the complementary nucleotides are contiguousnucleotides.

In certain embodiments, the siNA molecule portion of the myostatin siNAconjugates that are administered in vivo as per the methods of thepresent invention have between about 15 to about 30 (e.g., about 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotidesin the antisense strand that are complementary to a nucleotide sequenceof a myostatin target nucleic acid. In certain embodiments, the siNAmolecules of the invention comprise an antisense strand having at least15 nucleotides having sequence complementarity to a myostatin targetsequence. In certain embodiments, the siNA molecules of the inventioncomprise an antisense strand having at least 18 nucleotides havingsequence complementarity to a myostatin target sequence. In certainembodiments, the siNA molecules of the invention comprise an antisensestrand having at least 19 nucleotides having sequence complementarity toa myostatin target sequence. In certain embodiments, the siNA moleculesof the invention comprise an antisense strand having at least 20nucleotides having sequence complementarity to a myostatin targetsequence. In certain embodiments, the siNA molecules of the inventioncomprise an antisense strand having at least 21 nucleotides havingsequence complementarity to a myostatin target sequence. In certainembodiments of this aspect of the invention, the complementarynucleotides are contiguous nucleotides.

In some embodiments, a double-stranded siNA molecule comprised with themyostatin siNA conjugates described herein has perfect complementaritybetween the sense strand or sense region and the antisense strand orantisense region of the siNA molecule, with the exception of anyoverhanging region.

In yet other embodiments, a double-stranded siNA molecule has partialcomplementarity (i.e., less than 100% complementarity) between the sensestrand or sense region and the antisense strand or antisense region ofthe siNA molecule. Thus, in some embodiments, the double-strandednucleic acid molecules have between about 15 to about 30 (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides in one strand (e.g., sense strand) that are complementary tothe nucleotides of the other strand (e.g., antisense strand). In certainembodiments, the double-stranded siNA molecules have 17 nucleotides inthe sense region that are complementary to nucleotides of the antisenseregion of the molecule. In certain embodiments, the double-stranded siNAmolecules have 18 nucleotides in the sense region that are complementaryto nucleotides of the antisense region of the molecule. In certainembodiments, the double-stranded siNA molecules have 19 nucleotides inthe sense region that are complementary to nucleotides of the antisenseregion of the molecule. In certain embodiments, the double-stranded siNAmolecules of the invention have 20 nucleotides in the sense region thatare complementary to nucleotides of the antisense region of themolecule. In certain embodiments of this aspect of the invention, thecomplementary nucleotides between the strands are contiguousnucleotides.

For siNA molecules that are symmetric, each strand, the sense(passenger) strand and antisense (guide) strand, are independently about15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30) nucleotides in length. Generally, each strand ofa symmetric siNA molecule is about 19-24 (e.g., about 19, 20, 21, 22, 23or 24) nucleotides in length. In certain embodiments, each strand of asymmetric siNA molecule is 19 nucleotides in length. In certainembodiments, each strand of a symmetric siNA molecule is 20 nucleotidesin length. In certain embodiments, each strand of a symmetric siNAmolecule is 21 nucleotides in length. In certain embodiments, eachstrand of a symmetric siNA molecule is 22 nucleotides in length.

For siNA molecules that are asymmetric, the antisense strand of themolecule is about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length,wherein the sense region is about 3 to about 25 (e.g., about 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or25) nucleotides in length. Generally, the antisense strand of anasymmetric siNA molecule is about 19-24 (e.g., about 19, 20, 21, 22, 23or 24) nucleotides in length. In one embodiment, the sense strand of anasymmetric siNA molecule is about 19-24 (e.g., about 19, 20, 21, 22, 23or 24) nucleotides in length.

In yet other embodiments, siNA molecules comprised with the myostatinsiNA conjugates described herein are hairpin siNA molecules, wherein thesiNA molecules are about 25 to about 70 (e.g., about 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 40, 45, 50, 55, 60, 65, or 70) nucleotidesin length.

In certain embodiments, siNA molecules comprised with the myostatin siNAconjugates described herein are microRNA mimetics, having a nucleotidesequence comprising a nucleotide portion that is fully or partiallyidentical to a seed region of a myostatin-related microRNA. In certainembodiments, the nucleotide sequence of a microRNA mimetic has anucleotide portion that is 100% identical to a seed region of amyostatin-related microRNA. In certain embodiments, the nucleotidesequence of a microRNA mimetic has a nucleotide portion that is at least75% identical (e.g., about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or99%) to a seed region of a myostatin-related microRNA. In certainembodiments, the nucleotide sequence of a myostatin-related microRNAmimetic has a nucleotide portion that is 75% identical to a seed regionof a myostatin-related microRNA. In certain embodiments, the nucleotidesequence of a microRNA mimetic has a nucleotide portion that is 80%identical to a seed region of a myostatin-related microRNA. In certainembodiments, the nucleotide sequence of a microRNA mimetic has anucleotide portion that is 90% identical to a seed region of amyostatin-related microRNA. In certain embodiments, the nucleotidesequence of a microRNA mimetic has a nucleotide portion that is 95%identical to a seed region of a myostatin-related microRNA.

In other embodiments, siNA molecules comprised within the myostatin siNAconjugates described herein can contain one or more nucleotidedeletions, substitutions, mismatches and/or additions (in reference to amyostatin target site sequence, or between strands of a duplex siNAmolecule); provided, however, that the siNA molecule maintains itsactivity, for example, to mediate RNAi. In a non-limiting example, thedeletion, substitution, mismatch and/or addition can result in a loop orbulge, or alternately a wobble or other alternative (non Watson-Crick)base pair. Thus, in some embodiments, for example, double-strandednucleic acid siNA molecules have one or more (e.g., 1, 2, 3, 4, 5, or 6)nucleotides in one strand or region (e.g., sense strand) that aremismatches or non-base-paired with the other strand or region (e.g.,antisense strand). In certain embodiments, the siNA molecules contain nomore than 3 mismatches. If the antisense strand of an siNA moleculecontains mismatches to a myostatin target sequence, it is preferablethat the area of mismatch is not located in the center of a contiguousregion of complementarity.

In certain embodiments, the siNA molecules comprised with the conjugatesdescribed herein comprise overhangs of about 1 to about 4 (e.g., about1, 2, 3 or 4) nucleotides. The nucleotides in the overhangs can be thesame or different nucleotides. In some embodiments, the overhangs occurat the 3′-end (or the 3′ terminus) of one or both strands ofdouble-stranded siNA molecules. For example, a double-stranded siNAmolecule can comprise a nucleotide or non-nucleotide overhang at the3′-end of the antisense strand/region, at the 3′-end of the sensestrand/region, or at the 3′ ends of both the antisense strand/region andthe sense strand/region. Overhanging nucleotides can be modified orunmodified.

In some embodiments, the nucleotides comprising the overhanging portionof an siNA molecule comprise sequences based on a myostatin targetnucleic acid sequence in which the nucleotides comprising theoverhanging portion of the antisense strand/region are complementary tonucleotides in the myostatin target polynucleotide sequence and/or thenucleotides comprising the overhanging portion of the sensestrand/region comprise nucleotides from the myostatin targetpolynucleotide sequence. Thus, in some embodiments, the overhangcomprises a two nucleotide overhang that is complementary to a portionof the myostatin target polynucleotide sequence. In other embodiments,however, the overhang comprises a two nucleotide overhang that is notcomplementary to a portion of the myostatin target nucleic acidsequence. In certain embodiments, the overhang comprises a 3′-UUoverhang that is not complementary to a portion of the myostatin targetnucleic acid sequence. In other embodiments, the overhang comprises a UUoverhang at the 3′ end of the antisense strand and a TT overhang at the3′ end of the sense strand.

In any of the embodiments of the siNA molecules described herein having3′-end nucleotide overhangs, the overhangs are optionallychemically-modified at one or more nucleic acid sugar, base, or backbonepositions. Representative, but not limiting examples of modifiednucleotides in the overhanging portion of a double-stranded siNAmolecule include the following: 2′-O-alkyl (e.g., 2′-O-methyl),2′-deoxy, 2′-deoxy-2′-fluoro, 2′-deoxy-2′-fluoroarabino (FANA), 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,2′-O-difluoromethoxy-ethoxy, universal base, acyclic, or 5-C-methylnucleotides. In more preferred embodiments, the overhang nucleotides areeach independently, a 2′-O-alkyl nucleotide, a 2′-O-methyl nucleotide, a2′-deoxy-2-fluoro nucleotide, or a 2′-deoxy ribonucleotide. In someinstances the overhanging nucleotides are linked by one or morephosphorothioate linkages.

In yet other embodiments, siNA molecules comprised within the myostatinsiNA conjugates described herein comprise duplex nucleic acid moleculeswith blunt ends (i.e., without nucleotide overhangs), where both terminiof the molecule are blunt, or alternatively, where one of the ends isblunt. In some embodiments, the siNA molecules comprise one blunt end,for example wherein the 5′-end of the antisense strand and the 3′-end ofthe sense strand do not have any overhanging nucleotides, or wherein the3′-end of the antisense strand and the 5′-end of the sense strand do nothave any overhanging nucleotides. In other embodiments, siNA moleculescomprise two blunt ends, for example wherein the 3′-end of the antisensestrand and the 5′-end of the sense strand, as well as the 5′-end of theantisense strand and 3′-end of the sense strand, do not have anyoverhanging nucleotides.

In any of the embodiments or aspects of the siNA molecules comprisedwithin the myostatin siNA conjugates described herein, the sense strandand/or the antisense strand can further have a cap, such as describedherein or as known in the art. A cap can be present at the 3′-end of theantisense strand, the 5′-end of the sense strand, and/or the 3′-end ofthe sense strand. In the case of a hairpin siNA molecule, a cap can bepresent at the 3′-end of the polynucleotide. In some embodiments, a capis at one or both ends of the sense strand of a double-stranded siNAmolecule. In other embodiments, a cap is at the 3′-end of antisense(guide) strand. In other embodiments, a cap is at the 3′-end of thesense strand and at the 5′-end of the sense strand. Representative butnon-limiting examples of such terminal caps include an inverted abasicnucleotide and derivatives thereof, an inverted nucleotide moiety, aglyceryl modification, an alkyl or cycloalkyl group, a heterocycle orany other cap as is generally known in the art.

Any of the embodiments of the siNA molecules described herein can have a5′ phosphate terminus. In some embodiments, the siNA molecules lackterminal phosphates.

In certain embodiments, double-stranded siNA molecules comprised withinthe myostatin siNA conjugates described herein comprise about 3 to about30 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs.Generally, the duplex structure of siNA molecules is between 15 and 30base pairs, more generally between 18 and 25 base pairs, yet moregenerally between 19 and 24 base pairs, and most generally between 19and 21 base pairs in length. In one embodiment, a double-stranded siNAmolecule comprises 19 base pairs. In one embodiment, a double-strandedsiNA molecule comprises 20 base pairs. In one embodiment, adouble-stranded siNA molecule comprises 21 base pairs. Thedouble-stranded siNA molecules can be asymmetric or symmetric. In otherembodiments of this aspect of the invention, the siNA duplex moleculesare hairpin structures.

Any siNA molecule can comprise one or more chemically-modifiednucleotides. Modifications can be used to improve in vitro or in vivocharacteristics such as stability, activity, toxicity, immune response(e.g., prevent stimulation of an interferon response, an inflammatory orpro-inflammatory cytokine response, or a Toll-like Receptor response),and/or bioavailability. Various chemically modified siNA motifsdisclosed herein have the potential to maintain an RNAi activity that issubstantially similar to either unmodified or minimally-modified activesiRNA (see for example Elbashir et al., 2001, EMBO J., 20:6877-6888)while, at the same time, providing nuclease resistance andpharmacokinetic properties suitable for use in therapeutic applications.

In certain embodiments of the siNA molecules comprised with themyostatin siNA conjugates used by the methods of the present invention,any (e.g., one, more or all) nucleotides present in the antisense and/orsense strand may be modified nucleotides (e.g., wherein one nucleotideis modified, some nucleotides (i.e., a plurality or more than one) aremodified, or all nucleotides of the molecule are modified nucleotides).Modifications include sugar modifications, base modifications, backbone(internucleoside linkage) modifications, non-nucleotide modifications,and/or any combination thereof.

Non-limiting examples of chemical modifications that are suitable foruse in the siNA molecule portion of the conjugates described herein aredisclosed in U.S. Pat. No. 8,202,979 and U.S. patent application Ser.Nos. 10/981,966 and 12/064,014 (published as US 20050266422 and US20090176725, respectively), and in references cited therein, and includesugar, base, and backbone modifications, non-nucleotide modifications,and/or any combination thereof. These U.S. patents and applications areincorporated hereby as references for the purpose of describing chemicalmodifications that are suitable for use with the siNA molecules.

The chemical modifications of nucleotides present within a single siNAmolecule can be the same or different. In some embodiments, at least onestrand of an siNA molecule has at least one chemical modification. Inother embodiments, each strand has at least one chemical modification,which can be the same or different, such as sugar, base, or backbone(i.e., internucleotide linkage) modifications. In other embodiments,siNA molecules contain at least 2, 3, 4, 5, or more different chemicalmodifications.

In some embodiments, the siNA molecules comprised within the myostatinsiNA conjugates administered as per the methods of the present inventionare partially modified (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 55, or 59 nucleotides are modified) with chemicalmodifications. In some embodiments, an siNA molecule comprises at leastabout 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, or 60 nucleotides that are modifiednucleotides, excluding the 5′ modified nucleotide of the antisensestrand. In other embodiments, the siNA molecules are completely modified(100% modified) with chemical modifications, i.e., the siNA moleculedoes not contain any ribonucleotides. In some embodiments, one or moreof the nucleotides in the sense strand of the siNA molecules aremodified. In the same or other embodiments, one or more of thenucleotides in the antisense strand of the siNA molecules are modified,excluding the 5′ modified nucleotide of the antisense strand. In someembodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or30) of the nucleotide positions independently in either one or bothstrands of an siNA molecule are modified.

Modified nucleotides contained within the siNA molecules include thosewith modifications at the 2′-carbon of a sugar moiety and/or the3′-carbon of a sugar moiety of a nucleotide. In certain specificembodiments of the invention, at least one modified nucleotide is a2′-deoxy-2-fluoro nucleotide, a 2′-deoxy nucleotide, a 2′-O-alkyl (e.g.,2′-O-methyl) nucleotide, a 2′-methoxyethoxy or a locked nucleic acid(LNA) nucleotide, as is generally recognized in the art.

In yet other embodiment of the invention, at least one nucleotide has aribo-like, Northern or A form helix configuration (see e.g., Saenger,Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984).Non-limiting examples of nucleotides having a Northern configurationinclude locked nucleic acid (LNA) nucleotides (e.g., 2′-0,4′-C-methylene-(D-ribofuranosyl) nucleotides); 2′-methoxyethoxy (MOE)nucleotides; 2′-methyl-thio-ethyl nucleotides; 2′-deoxy-2′-fluoronucleotides; 2′-deoxy-2′-chloro nucleotides; 2′-azido nucleotides;2′-O-trifluoromethyl nucleotides; 2′-O-ethyl-trifluoromethoxynucleotides; 2′-O-difluoromethoxy-ethoxy nucleotides; 4′-thionucleotides; and 2′-O-methyl nucleotides. In various embodiments, amajority (e.g., greater than 50%) of the pyrimidine nucleotides presentin a double-stranded siNA molecule comprises a sugar modification. Insome of the same and/or other embodiments, a majority (e.g., greaterthan 50%) of the purine nucleotides present in a double-stranded siNAmolecule comprises a sugar modification.

In certain instances, purine and pyrimidine nucleotides of an siNAmolecule are differentially modified. In one example, purine andpyrimidine nucleotides can be differentially modified at the 2′-carbonof the sugar moiety (i.e., at least one purine has a differentmodification from at least one pyrimidine in the same or differentstrand at the 2′-carbon of the sugar moiety). In certain embodiments,the purines are unmodified in one or both strands, while the pyrimidinesin one or both strands are modified. In certain other instances, thepyrimidines are unmodified in one or both strands, while the purines inone or both strands are modified. In certain instances, wherein the siNAmolecules comprise one or more modifications as described herein, thenucleotides at positions 2 and 3 at the 5′ end of the antisense (guide)strand are unmodified.

In some embodiments of the siNA molecules, the pyrimidine nucleotides inthe antisense strand are 2′-O-methyl or 2′-deoxy-2′-fluoro pyrimidinenucleotides, and the purine nucleotides present in the antisense strandare 2′-O-methyl nucleotides or 2′-deoxy nucleotides. In certainembodiments, all of the pyrimidine nucleotides in a complementary regionof an antisense strand of an siNA molecule are 2′-deoxy-2′-fluoropyrimidine nucleotides. In certain embodiments, all of the purines inthe complementary region on the antisense strand are 2′-O-methyl purinenucleotides.

In other embodiments of the siNA molecules, the pyrimidine nucleotidesin the sense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides, andthe purine nucleotides present in the sense strand are 2′-O-methyl or2′-deoxy purine nucleotides. In certain embodiments of the invention,all the pyrimidine nucleotides in the complementary region on the sensestrand are 2′-deoxy-2′-fluoro pyrimidine nucleotides. In certainembodiments, all the purine nucleotides in the complementary region onthe sense strand are 2′-deoxy purine nucleotides.

In certain embodiments, all of the pyrimidine nucleotides in thecomplementary regions on the sense strand are 2′-deoxy-2′-fluoropyrimidine nucleotides; all of the pyrimidine nucleotides in thecomplementary region of the antisense strand are 2′-deoxy-2′-fluoropyrimidine nucleotides; all the purine nucleotides in the complementaryregion on the sense strand are 2′-deoxy purine nucleotides and all ofthe purines in the complementary region on the antisense strand are2′-O-methyl purine nucleotides.

In some embodiments, at least 5 or more of the pyrimidine nucleotides inone or both strands of an siNA molecule are 2′-deoxy-2′-fluoropyrimidine nucleotides. In some embodiments, at least 5 or more of thepyrimidine nucleotides in one or both strands are 2′-O-methyl pyrimidinenucleotides. In some embodiments, at least 5 or more of the purinenucleotides in one or both strands are 2′-deoxy-2′-fluoro purinenucleotides In some embodiments, at least 5 or more of the purinenucleotides in one or both strands are 2′-O-methyl purine nucleotides.

In certain embodiments, the siNA molecules comprise one or more modifiedinternucleoside linking group. A modified internucleoside linking groupis a linking group other than a phosphodiester 3′-5′ internucleosidelinking group, including but not limited to 2′ internucleoside linkinggroups (e.g., phosphodiester and phosphorothioate 2′-5′ internucleosidelinkages). In certain embodiments, each internucleoside linking groupis, independently, a 2′ or 3′ phosphodiester or phosphorothioateinternucleoside linking group. In certain embodiments, the 5′-mostinternucleoside linking group on either or both strands of an siNAmolecule is a phosphorothioate linking group. In certain embodiments,the siNA molecules comprise from 3 to 12 contiguous phosphorothioatelinking groups, wherein the phosphorothioate linking groups are either2′ or 3′ internucleoside linking groups. In certain embodiments, thesiNA molecules comprise from 6 to 8 contiguous phosphorothioate linkinggroups, wherein the phosphorothioate linking groups are either 2′ or 3′internucleoside linking groups. In certain embodiments, the 3′ end ofthe antisense and/or sense strand of the siNA molecules comprises aphosphorothioate linking groups. In certain embodiments, the siNAmolecules comprise from 6 to 8 contiguous phosphorothioate linkinggroups at the 3′ end of the antisense and/or sense strand, wherein thephosphorothioate linking groups are either 2′ or 3′ internucleosidelinking groups.

Any of the above described modifications, or combinations thereof,including those in the references cited, can be applied to any of thesiNA molecules comprised within the myostatin siNA conjugates that areadministered as per the methods and uses of the present invention.

The myostatin siNA molecules can be obtained using a number oftechniques known to those of skill in the art. For example the siNAmolecules can be chemically synthesized using protocols known in the art(for example, as described in: Caruthers et al., 1992, Methods inEnzymology 211, 3-19; Thompson et al., International PCT Publication No.WO 99/54459; Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684;Wincott et al., 1997, Methods Mol. Bio., 74, 59; Brennan et al., 1998,Biotechnol Bioeng., 61, 33-45; Brennan, U.S. Pat. No. 6,001,311; Usmanet al., 1987, J. Am. Chem. Soc., 109, 7845; and Scaringe et al., 1990,Nucleic Acids Res., 18, 5433). The syntheses of oligonucleotidesdescribed in the art makes use of common nucleic acid protecting andcoupling groups, such as dimethoxytrityl at the 5′-end andphosphoramidites at the 3′- or 2′-end.

In certain embodiments, the siNA molecules are synthesized, deprotected,and analyzed according to methods described in, for example, U.S. Pat.Nos. 6,995,259, 6,686,463, 6,673,918, 6,649,751, 6,989,442, and7,205,399. In a non-limiting synthesis example, small scale synthesesare conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2μmol scale protocol with a 2.5 min coupling step for 2′-O-methylatednucleotides and a 45 second coupling step for 2′-deoxy nucleotides or2′-deoxy-2′-fluoro nucleotides.

Alternatively, the siNA molecules can be synthesized separately andjoined together post-synthetically, for example, by ligation (e.g.,Moore et al., 1992, Science 256, 9923; Draper et al., International PCTPublication No. WO 93/23569; Shabarova et al., 1991, Nucleic AcidsResearch 19, 4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16,951; and Bellon et al., 1997, Bioconjugate Chem. 8, 204), or byhybridization following synthesis and/or deprotection.

C. Myostatin siNA Conjugates

The present invention provides myostatin siNA molecules capable ofmediating RNA interference and reducing the in vivo expression ofmyostatin and methods for delivering the same to a subject, wherein thesiNA molecules that are delivered as per the methods disclosed hereinare linked to a lipophilic moiety, such as cholesterol. The lipophilicmoiety-linked myostatin siNA molecules are referred to herein asmyostatin siNA conjugates. In one embodiment, the myostatin siNAconjugates delivered by the methods of the present invention are notformulated within lipid formulations that form liposomes (e.g., a lipidnanoparticle). While not wishing to be bound by any particular theory,it is believed the attachment of a lipophilic moiety increases thelipophilicity of the myostatin siNA molecule, enhancing the entry of thesiNA molecule into muscle cells.

Examples of lipophilic moieties that can be linked to a myostatin siNAmolecule to form a myostatin siNA conjugate include, but are not limitedto cholesterol, oleic acid, stearic acid, palmitic acid, myristic acid,linoleic acid, oleyl, retinyl, cholesteryl residues, cholic acid,adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl,or phenoxazine. In a preferred embodiment, the linked lipophilic moietyis cholesterol.

In certain embodiments, the lipophilic moiety is attached directly tothe siNA molecule. In these embodiments, the lipophilic moiety is stillconsidered, for the purposes of the present invention, to be “linked” or“conjugated” to the siNA molecule. In certain embodiments, thelipophilic moiety is attached to the siNA molecule by means of aconventional linker or spacer molecule. The linker or spacer can be anucleic acid or non-nucleic acid linker/spacer. A number of linkermolecules are commercially available. Suitable linkers include, but arenot limited to, straight or branched-chain carbon linkers, heterocycliccarbon linkers, or peptide linkers. Although a linker or spacer moleculegenerally has no specific biological activity other than to join themolecules being combined, or to preserve some minimum distance or otherspatial relationship between them, the constituent amino acids of apeptide spacer may be selected to influence some property of themolecule such as the folding, net charge, or hydrophobicity.

The linker/spacer can be a nucleic acid linker that is biodegradable.The stability of a nucleic acid-based biodegradable linker molecule canbe modulated by using various chemistries, for example combinations ofribonucleotides, deoxyribonucleotides, and chemically-modifiednucleotides, such as 2′-O-methyl, 2′-fluoro, 2′-amino, 2′-O-amino,2′-C-allyl, 2′-O-allyl, and other 2′-modified or base modifiednucleotides. A biodegradable nucleic acid linker molecule can be adimer, trimer, tetramer or longer nucleic acid molecule, for example, anoligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 nucleotides in length, or can comprise a singlenucleotide with a phosphorus-based linkage, for example, aphosphoramidite or phosphodiester linkage. A biodegradable nucleic acidlinker molecule can also comprise nucleic acid backbone, nucleic acidsugar, or nucleic acid base modifications.

The lipophilic moiety is attached to a myostatin siNA molecule throughattachment to either a terminus of the siNA molecule (e.g., the 3′ or 5′end of an oligonucleotide strand of the siNA molecule) or throughlinkage to an internal nucleotide of the siNA molecule. In oneembodiment, the lipophilic moiety is attached to the 3′ end of thepassenger strand (sense strand) of a double-stranded myostatin siNAmolecule. In one embodiment, the lipophilic moiety is attached to the 5′end of the passenger strand of a double-stranded myostatin siNAmolecule. In a further embodiment, the lipophilic moiety is attached tothe 3′ end of the guide strand (antisense strand) of a myostatin siNAmolecule. In a further embodiment, the lipophilic moiety is attached tothe 5′ end of the guide strand (antisense strand) of a myostatin siNAmolecule. In a further embodiment, a myostatin siNA conjugate containsmore than one attached lipophilic moiety (e.g., a lipophilic moietyattached to both the 3′ and the 5′ end of the passenger strand; alipophilic moiety attached to the 3′ end of the guide strand and the 5′end of the passenger strand). In this aspect of the invention, thelipophilic moieties can be the same or different.

In certain embodiments, a myostatin siNA conjugate is prepared bychemically conjugating all or a portion of a myostatin siNA molecule tothe lipophilic group. Means of chemically conjugating molecules are wellknown to those of skill in the art. Such means will vary according tothe structure of the moiety to be attached, but will be readilyascertainable to those of skill in the art.

The present invention further provides myostatin siNA conjugates in kitform. The kit may comprise a container. In one embodiment, the kitcontains one or more myostatin siNA conjugate with instructions forsystemic administration. The kits may comprise a myostatin siNAconjugate within a pharmaceutically acceptable carrier or diluent. Thekits may further comprise excipients.

D. Uses

The methods for systemically administering myostatin siNA conjugatesdescribed herein are useful to modulate and or regulate (e.g., inhibit,down-regulate) the in vivo expression and/or activity of a myostatintarget nucleic acid (e.g., a myostatin target gene) by an RNAiinterference mechanism (e.g., by degrading a myostatin mRNA). Modulationof the in vivo expression of a myostatin target nucleic acid resultsincreased muscle mass and/or enhanced muscle performance. The methodsmay be further useful in therapeutic regimens to treat one or moremusculoskeletal disease states. In one embodiment, inhibition of adisease may be evaluated by directly measuring the progress of thedisease in a subject. It may also be inferred through observing a changeor reversal in a condition associated with the disease. The methods ofthe present invention have the further potential of being used as aprophylaxis. Thus, use of the myostatin siNA conjugates andpharmaceutical compositions described herein have the potential ofameliorating, treating, preventing, and/or curing diseases statesassociated with regulation of myostatin gene expression. The myostatinsiNA conjugates further have the potential for use in cosmeticapplications and/or for veterinary purposes to increase muscle massand/or enhance muscle performance.

In an embodiment of the invention, the subject to which a myostatin siNAconjugate described herein is systemically administered is sufferingfrom a musculoskeletal disease or disorder. In one embodiment, amusculoskeletal disease or disorder includes a condition that causes orresults in muscle atrophy. Muscle atrophy can result from treatment witha glucocorticoid such as cortisol, dexamethasone, betamethasone,prednisone, methylprednisolone or prednisolone. Muscle atrophy can alsobe a result of denervation due to nerve trauma or a result ofdegenerative, metabolic or inflammatory neuropathy. For example, muscleatrophy can be a result of an adult motor neuron disease, Guillian-Barrésyndrome, infantile spinal muscular atrophy, amyotrophic lateralsclerosis, juvenile spinal muscular atrophy, autoimmune motor neuropathywith multifocal conductor block, paralysis due to stroke or spinal cordinjury, skeletal immobilization due to trauma, prolonged bed rest,voluntary inactivity, involuntary inactivity, and metabolic stress ornutritional insufficiency. Muscle atrophy can be a result of myopathy,including for example myotonia; a congenital myopathy, includingnemalene myopathy, multi/minicore myopathy and myotubular(centronuclear) myopathy; mitochondrial myopathy; familial periodicparalysis; inflammatory myopathy; metabolic myopathy, such as caused bya glycogen or lipid storage disease; dermatomyositis; polymyositis;inclusion body myositis; myositis ossificans; rhabdomyolysis andmyoglobinurias. Myopathy may be caused by a muscular dystrophy syndrome,such as Duchenne muscular dystrophy (DMD), Becker muscular dystrophy(also known as benign pseudohypertrophic muscular dystrophy), myotonicdystrophy, scapulohumeral and fascioscapulohumeral muscular dystrophy,Emery-Dreifuss muscular dystrophy, oculopharyngeal muscular dystrophy,limb girdle muscular dystrophy, Fukuyama congenital muscular dystrophy,or hereditary distal myopathy.

Further examples musculoskeletal disease or disorder or conditions thatresult in musculoskeletal disease or disorder include sarcopenia, skinatrophy, muscle wasting, brain atrophy, atherosclerosis,arteriosclerosis, pulmonary emphysema, osteoporosis, osteoarthritis,immunologic incompetence, high blood pressure, dementia, Huntington'sdisease, Alzheimer's disease, cataracts, age-related maculardegeneration, cancer, stroke, frailty, memory loss, impaired kidneyfunction, metabolic disorders (including Type-II diabetes, metabolicsyndrome, hyperglycemia, obesity, thyroid gland disorder), cachexia(including cachexia associated with a rheumatoid arthritis and cachexiaassociated with cancer), acute and/or chronic renal disease or failure,liver diseases (examples such as fibrosis, cirrhosis), cancer (includingrhabdomyosarcoma, prostate cancer, breast cancer, hepatocellularcarcinoma, and gastrointestinal cancer), Parkinson's Disease; anemia,exposure to environmental toxins or drugs, HIV/AIDS, fasting, benigncongenital hypotonia, central core disease, burn injury, chronicobstructive pulmonary disease, sepsis, congestive heart failure, agingor an age-related condition, and space travel or time spent in a zerogravity environment.

The myostatin siNA conjugates and pharmaceutical formulations thereofcan be administered to a subject alone or used in combination with oneor more other therapies, including known therapeutic agents, treatments,or procedures to prevent or treat musculoskeletal diseases, disorders,conditions, and traits. A combination can conveniently be presented foruse in the form of a pharmaceutical composition, wherein thepharmaceutical composition comprises a combination that includes amyostatin siNA conjugate, a pharmaceutically acceptable diluent orcarrier, and one or more additional therapeutic agents. Alternatively,the individual components of such combinations can be administeredeither sequentially or simultaneously in separate or combinedpharmaceutical formulations.

Combinations of the methods of the invention with standard medicaltreatments (e.g., corticosteroids for muscular dystrophies) arespecifically contemplated, as are combinations with novel therapies. Forexample, for treatment of genetic muscular dystrophies, methods of theinvention may be combined with follistatin administration, followed bysimultaneous or concomitant treatment to correct the genetic disorder.Correcting a genetic disorder may involve, for example, replacingsarcoglycans in sarcoglycan deficiency, correcting or replacingdystrophin in disorders such as Duchenne's Muscular Dystrophy, treatingALS patients with IGF-1 or mutant SOD1 interference strategies. Giventhat in a disorder contemplated for treatment by the methods of thepresent invention, a significant amount of muscle may be lost, therescue of muscle will provide a substrate (preserved or regeneratedmuscle) for subsequent gene correction. In this respect, it may beconceivable to inhibit myostatin to enhance muscle, increase musclesize, and then provide the secondary treatment. Such secondarytreatments for muscular dystrophy may be IGF-1, exon-skipping, calpaininhibition, dystrophin upregulation, and dystroglycan expression.Myostatin inhibition in concert with muscle precursor cells (satellitecells, stem cells) may allow more of these cells to be incorporated intothe tissue.

E. Pharmaceutical Compositions

The myostatin siNA conjugates of the invention are preferably formulatedas pharmaceutical compositions prior to systemically administering to asubject, according to techniques known in the art. Pharmaceuticalcompositions are characterized as being at least sterile andpyrogen-free. Methods for preparing pharmaceutical compositions arewithin the skill in the art for example as described in Remington'sPharmaceutical Science, 17^(th) ed., Mack Publishing Company, Easton,Pa. (1985).

Pharmaceutical compositions of the myostatin siNA conjugates furthercomprise conventional pharmaceutical excipients and/or additives.Suitable pharmaceutical excipients include preservatives, flavoringagents, stabilizers, antioxidants, osmolality adjusting agents, buffers,and pH adjusting agents. Suitable additives include physiologicallybiocompatible buffers (e.g., trimethylamine hydrochloride), addition ofchelants (such as, e.g., DTPA or DTPA-bisamide) or calcium chelatecomplexes (e.g. calcium DTPA, CaNaDTPA-bisamide), or, optionally,additions of calcium or sodium salts (e.g., calcium chloride, calciumascorbate, calcium gluconate or calcium lactate). In addition,antioxidants and suspending agents can be used.

Compositions intended for oral use may be prepared according to methodsknown in the art for the manufacture of pharmaceutical compositions,especially methods known in the art for the manufacture ofpharmaceutical compositions comprising oligonucleotides. For example,oral delivery of siRNA and antisense oligonucleotides has been achievedthrough encapsulating siRNA within biodegradable particles that protectthem from degradation and target them to M cells in intestinal Peyer'spatches (see Akhtar, S., 2009, J. Drug Target. 17:491-495). Oralcomposition can contain one or more such sweetening agents, flavoringagents, coloring agents or preservative agents in order to providepharmaceutically elegant and palatable preparations. Tablets contain theactive ingredient in admixture with non-toxic pharmaceuticallyacceptable excipients that are suitable for the manufacture of tablets.These excipients can be, e.g., inert diluents (such as calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate), granulating and disintegrating agents (e.g., corn starch, oralginic acid), binding agents (e.g., starch, gelatin or acacia), andlubricating agents (e.g., magnesium stearate, stearic acid or talc). Thetablets can be uncoated or they can be coated by known techniques. Insome cases such coatings can be prepared by known techniques to delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a timedelay material such as glyceryl monosterate or glyceryl distearate canbe employed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent,e.g., calcium carbonate, calcium phosphate or kaolin, or as soft gelatincapsules wherein the active ingredient is mixed with water or an oilmedium, e.g., peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in a mixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, e.g., sodium carboxymethylcellulose,methylcellulose, hydropropyl-methylcellulose, sodium alginate,polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing orwetting agents can be a naturally-occurring phosphatide, e.g., lecithin,or condensation products of an alkylene oxide with fatty acids, e.g.,polyoxyethylene stearate; or condensation products of ethylene oxidewith long chain aliphatic alcohols, e.g., heptadecaethyleneoxycetanol,or condensation products of ethylene oxide with partial esters derivedfrom fatty acids and a hexitol such as polyoxyethylene sorbitolmonooleate, or condensation products of ethylene oxide with partialesters derived from fatty acids and hexitol anhydrides, e.g.,polyethylene sorbitan monooleate. The aqueous suspensions can alsocontain one or more preservatives, e.g. ethyl, or n-propylp-hydroxybenzoate, one or more coloring agents, one or more flavoringagents, and one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, e.g. arachis oil, olive oil, sesame oil or coconutoil, or in a mineral oil such as liquid paraffin. The oily suspensionscan contain a thickening agent, e.g. beeswax, hard paraffin or cetylalcohol. Sweetening agents and flavoring agents can be added to providepalatable oral preparations. These compositions can be preserved by theaddition of an anti-oxidant such as ascorbic acid

Pharmaceutical compositions of the invention can also be in the form ofoil-in-water emulsions. The oily phase can be a vegetable oil or amineral oil or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, e.g. gum acacia or gum tragacanth,naturally-occurring phosphatides, e.g. soy bean, lecithin, and esters orpartial esters derived from fatty acids and hexitol, anhydrides, forexample sorbitan monooleate, and condensation products of the saidpartial esters with ethylene oxide, e.g. polyoxyethylene sorbitanmonooleate. The emulsions can also contain sweetening and flavoringagents.

Syrups and elixirs can be formulated with sweetening agents, e.g.glycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations can also contain a demulcent, a preservative, and flavoringand coloring agents. The pharmaceutical compositions can be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension can be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation can also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilcan be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

The myostatin siNA conjugates can take the form of suppositories, e.g.,for rectal administration of the drug. These compositions can beprepared by mixing the drug with a suitable non-irritating excipientthat is solid at ordinary temperatures but liquid at the rectaltemperature and will therefore melt in the rectum to release the drug.Such materials include cocoa butter and polyethylene glycols.

Myostatin siNA conjugates described herein can be formulated in asterile medium for intravenous administration. The molecule, dependingon the vehicle and concentration used, can either be suspended ordissolved in the vehicle. Advantageously, adjuvants such as localanesthetics, preservatives and buffering agents can be dissolved in thevehicle.

In other embodiments, myostatin siNA conjugate formulations providedherein for use in pulmonary delivery further comprise one or moresurfactants. Suitable surfactants or surfactant components for enhancingthe uptake of the compositions of the invention include synthetic andnatural as well as full and truncated forms of surfactant protein A,surfactant protein B, surfactant protein C, surfactant protein D andsurfactant Protein E, di-saturated phosphatidylcholine (other thandipalmitoyl), dipalmitoylphosphatidylcholine, phosphatidylcholine,phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine,phosphatidylserine; phosphatidic acid, ubiquinones,lysophosphatidylethanolamine, lysophosphatidylcholine,palmitoyl-lysophosphatidylcholine, dehydroepiandrosterone, dolichols,sulfatidic acid, glycerol-3-phosphate, dihydroxyacetone phosphate,glycerol, glycero-3-phosphocholine, dihydroxyacetone, palmitate,cytidine diphosphate (CDP) diacylglycerol, CDP choline, choline, and/orcholine phosphate; as well as natural and artificial lamellar bodieswhich are the natural carrier vehicles for the components of surfactant,omega-3 fatty acids, polyenic acid, polyenoic acid, lecithin, palmitinicacid, non-ionic block copolymers of ethylene or propylene oxides,polyoxypropylene, monomeric and polymeric, polyoxyethylene, monomericand polymeric, poly (vinyl amine) with dextran and/or alkanoyl sidechains, Brij 35, Triton X-100 and synthetic surfactants ALEC, Exosurf,Survan and Atovaquone, among others. These surfactants can be usedeither as single or part of a multiple component surfactant in aformulation, or as covalently bound additions to the 5′ and/or 3′ endsof the nucleic acid component of a pharmaceutical composition herein.

In one embodiment, myostatin siNA conjugates can be formulated foradministration via pulmonary delivery, such as by inhalation of anaerosol or spray dried formulation administered by an inhalation deviceor nebulizer, providing rapid local uptake of the nucleic acid moleculesinto relevant pulmonary tissues. Solid particulate compositionscontaining respirable dry particles of micronized nucleic acidcompositions can be prepared by grinding dried or lyophilized nucleicacid compositions, and then passing the micronized composition through,for example, a 400 mesh screen to break up or separate out largeagglomerates. A solid particulate composition comprising the siNAconjugate compositions can optionally contain a dispersant which servesto facilitate the formation of an aerosol as well as other therapeuticcompounds. A suitable dispersant is lactose, which can be blended withthe nucleic acid compound in any suitable ratio, such as a 1 to 1 ratioby weight.

Spray compositions comprising siNA conjugates described herein can, forexample, be formulated as aqueous solutions or suspensions or asaerosols delivered from pressurized packs, such as a metered doseinhaler, with the use of a suitable liquefied propellant. In oneembodiment, aerosol compositions suitable for inhalation can be either asuspension or a solution and generally contain an siNA conjugate and asuitable propellant such as a fluorocarbon or hydrogen-containingchlorofluorocarbon or mixtures thereof, particularly hydrofluoroalkanes,especially 1,1,1,2-tetrafluoroethane,1,1,1,2,3,3,3-heptafluoro-n-propane or a mixture thereof. The aerosolcomposition can optionally contain additional formulation excipientswell known in the art such as surfactants. Non-limiting examples includeoleic acid, lecithin or an oligolactic acid or derivative such as thosedescribed in WO94/21229 and WO98/34596 and co-solvents for exampleethanol. In one embodiment, a pharmaceutical aerosol formulationcomprises an siNA conjugate and a fluorocarbon or hydrogen-containingchlorofluorocarbon or mixtures thereof as propellant, optionally incombination with a surfactant and/or a co-solvent.

The aerosol formulations can be buffered by the addition of suitablebuffering agents.

Aerosol formulations can include optional additives includingpreservatives if the formulation is not prepared sterile. Non-limitingexamples include, methyl hydroxybenzoate, anti-oxidants, flavorings,volatile oils, buffering agents and emulsifiers and other formulationsurfactants. In one embodiment, fluorocarbon or perfluorocarbon carriersare used to reduce degradation and provide safer biocompatiblenon-liquid particulate suspension compositions. In another embodiment, adevice comprising a nebulizer delivers an siNA conjugate compositioncomprising fluorochemicals that are bacteriostatic thereby decreasingthe potential for microbial growth in compatible devices.

Capsules and cartridges comprising the myostatin siNA conjugatecompositions for use in an inhaler or insufflator, of for examplegelatin, can be formulated containing a powder mix for inhalation of acompound of the invention and a suitable powder base such as lactose orstarch. In one embodiment, each capsule or cartridge contains an siNAconjugate and one or more excipients. In another embodiment, an siNAconjugate can be presented without excipients such as lactose.

The siNA conjugates can also be formulated as a fluid formulation fordelivery from a fluid dispenser, such as those described and illustratedin WO05/044354.

F. Administration

The myostatin siNA conjugates and pharmaceutical compositions thereofare introduced into a subject by any of a variety of forms of systemicadministration. For the purposes of the present invention, systemicadministration include pulmonary (inhalation, nebulization etc.),intravenous, subcutaneous, catheterization, nasopharyngeal, ororal/gastrointestinal administration as is generally known in the art.Further non-limiting examples of administration methods of the inventioninclude buccal, sublingual, parenteral (i.e., intravenously,intraperitoneally, subcutaneously, or intramuscularly), local rectaladministration or other local administration resulting in absorption oraccumulation of the myostatin siNA conjugates in the blood streamfollowed by distribution throughout the entire body. In one embodiment,the myostatin siNA conjugates and pharmaceutical compositions thereofcan be administered by insufflation and inhalation. In one embodiment,the myostatin siNA conjugates and pharmaceutical compositions thereofare administered intravenously or intraperitoneally by a bolus injection(see, e.g., U.S. Pat. No. 5,286,634). In another embodiment, themyostatin siNA conjugates and pharmaceutical compositions thereof areadministered subcutaneously. In a further embodiment, the myostatin siNAconjugates and pharmaceutical compositions thereof are administered inovo to an avian embryo while contained in the egg. The siNA conjugatesmay be administered to any suitable compartment of the egg (e.g.,allantois fluid, yolk sac, amnion, air cell or into the embryo).

For therapeutic applications, a pharmaceutically effective dose of themyostatin siNA conjugates or pharmaceutical compositions is systemicallyadministered to a subject. A pharmaceutically effective dose is thatdose required to prevent, inhibit the occurrence, or treat (alleviate asymptom to some extent, preferably all of the symptoms) a disease state.One skilled in the art can readily determine a therapeutically effectivedose of a myostatin siNA conjugate to be systemically administered to agiven subject, e.g., by taking into account factors, such as the sizeand weight of the subject, the extent of the disease progression orpenetration, the age, health, and sex of the subject, and the route ofsystemic administration. Generally, an amount between 0.1 μg/kg and 100mg/kg body weight/day of active ingredients is administered dependentupon potency of the negatively charged polymer. Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient. The myostatin siNA conjugates can be administered in asingle dose or in multiple doses.

In one embodiment, the siNA conjugates described herein are systemicallydelivered to a subject at a dose of between about 0.1 to about 500 mg/kg(mpk). In another embodiment, the siNA conjugates are delivered at adose of between about 1 to about 200 mpk. In another embodiment, thesiNA conjugates are delivered at a dose of between about 1 to about 100mpk. In another embodiment, the siNA conjugates are delivered at a doseof between about 5 to about 60 mpk. In another embodiment, the siNAconjugates are delivered at a dose of between about 10 to about 50 mpk.

The myostatin siNA conjugates can be administered once monthly, onceweekly, once daily (QD), or divided into multiple monthly, weekly, ordaily doses, such as, for example, twice daily (BID), three times daily(TID), once every two weeks. Thus, administration can be accomplishedvia single or divided doses. Persons of ordinary skill in the art caneasily estimate repetition rates for dosing based on measured residencetimes and concentrations of the drug in bodily fluids or tissues.

In addition, the administration can be continuous, e.g., every day, orintermittently. For example, intermittent administration of a myostatinsiNA conjugate may be administration one to six days per week,administration in cycles (e.g., daily administration for two to eightconsecutive weeks, then a rest period with no administration for up toone week), or administration on alternate days.

Aerosol compositions can be administered into the respiratory system asa formulation that includes particles of respirable size, e.g. particlesof a size sufficiently small to pass through the nose, mouth and larynxupon inhalation and through the bronchi and alveoli of the lungs. Ingeneral, respirable particles range from about 0.5 to 10 microns insize. In one embodiment, the particulate range can be from 1 to 5microns. In another embodiment, the particulate range can be from 2 to 3microns. Particles of non-respirable size which are included in theaerosol tend to deposit in the throat and be swallowed, and the quantityof non-respirable particles in the aerosol is thus minimized. For nasaladministration, a particle size in the range of 10-500 um is preferredto ensure retention in the nasal cavity.

EXAMPLES

Examples provided are intended to assist in a further understanding ofthe invention. Particular materials employed, species and conditions areintended to be illustrative of the invention and not limiting of thereasonable scope thereof. Certain starting materials and reagents areeither commercially available or known in the chemical scientific orpatent literature.

Example 1 Mtsn siRNA

siRNA Synthesis—

siRNAs were synthesized by methods similar to those previously described(Wincott, F. et al., 1995, Nucleic Acids Res. 23:2677-84). For eacholigonucleotide duplex, the individual, complementary sense andantisense strands were first synthesized on solid support, such as oncontrolled pore glass, using commercially available automatedoligosynthesizers. The solid support was obtained pre-loaded with thefirst (3′) nucleotide unit of the desired sequence and placed in anappropriate column for the oligosynthesizer. The first nucleotide waslinked to the solid support via a succinate linkage and contained asuitable acid sensitive protecting group (trityl, dimethoxytrityl) onthe 5′-terminal hydroxyl group. The solid-phase oligosynthesis employedsynthetic procedures that are generally known in the art. Elongation ofthe desired oligomeric sequence went through a cycle of four steps: 1)Acidic deprotection of the 5′-trityl protecting group; 2) Coupling ofthe next nucleotide unit as the 5′-trityl (or dimethoxytrityl) protectedphosphoramidite in the presence of an activating agent, such asS-ethyl-tetrazole; 3) Oxidation of the P(III) phosphite triester to theP(V) phosphate triester by an oxidizing agent, such as iodine; and 4)Capping any remaining unreacted alcohol groups through esterificationwith an acylating agent, such as acetic anhydride. The phosphoramiditesused were either derived from naturally occurring nucleotide units orfrom chemical modified versions of these nucleotides. Oligonucleotidesynthesis cycles were continued until the last (5′) nucleotide unit wasinstalled onto the extended oligomer. After the final cycle, the5′-trityl protecting group may or may not be removed from theoligonucleotide while it remains on the solid support. In someinstances, the 5′-terminal trityl was first removed by treatment with anacidic solution.

After deprotection, the solid support was treated with an appropriatebase, such as aqueous methylamine, in order to cleave theoligonucleotide from the support, remove the cyanoethyl protectinggroups on the phosphates and deprotect the acyl protecting groups on thenucleotide bases. After cleavage of each strand from the solid support,each strand was purified chromatographically with a reversed phase (C18)or anion exchange (SAX) resin. Typically, the oligonucleotide was elutedfrom SAX resin with a gradient of an inorganic salt, such as sodiumchloride. Salt was removed from the purified samples by dialysis ortangential flow filtration.

Each purified oligonucleotide was analyzed for purity by appropriatemethods, including reversed phase HPLC, SAX HPLC, and capillary gelelectrophoresis. The identity of the oligonucleotide was confirmed bymass spectrometry, using an ionization technique such as ESI or MALDI.The yields of each oligonucleotide were assessed by UV (260 nm) with atheoretically derived extinction coefficient.

The corresponding sense and antisense strands were annealed by mixing anequimolar amount of each material. The appropriate amounts of eachstrand were approximated by UV (260 nm) measurements and theoreticalextinction coefficients. After the annealing process, the extent ofduplex formation and the presence of any excess single strand materialwere assessed by an appropriate chromatographic method, such as RP-HPLCor SAX. When appropriate, the sample was adjusted with additionalamounts of one of the two strands in order to completely anneal theremaining excess single strand. The final duplex material waslyophilized prior to delivery for further biochemical or biologicaltesting.

Cells and Reagents—

Mouse hepatoma Hepa 1-6 cell line was obtained from the American TypeTissue Collection (Cat # CRL-1830). Cells were grown in Dulbecco'sModified Eagle Medium, High Glucose with Glutamax™ (Invitrogen Cat#10566024) adjusted to 1 mM sodium pyruvate (Invitrogen Cat #11360070and supplemented with 10% fetal bovine serum. Streptomycin andpenicillin were added to the media at 100 μg/mL and 100 U/mL,respectively. Cells were cultured at 37° C. in the presence of 5% CO₂.

Generation of Luciferase Reporter Constructs—

siRNAs were screened using the psiCHECK2 dual luciferase reportersystem. The luciferase reporter plasmids used were derived frompsiCHECK2 vector (Promega, Cat# C8021). Through de novo synthesis, thefull length transcript of mouse myostatin (NCBI GenBank RefSeqNM_010834) was cloned into the XhoI/NotI sites of the vector.

In Vitro Screening of siRNAs—

Hepa1-6 cells were seeded in 96-well plates at a density of 10,000 cellsper well and incubated at 37° C. After 24 hours, the cells wereco-transfected with siRNAs and the MSTN luciferase reporter plasmidusing Lipofectamine 2000 reagent (Life Technologies, Cat#11668019).Primary screens were performed by co-transfecting siRNAs with the MSTNplasmid at final concentrations of 10 nM and 0.6 ng/μL, respectively.The cells were incubated at 37° C. and culture medium was replaced withfresh media 24 hours post-transfection. After an additional 24 hourincubation, reporter Renilla luciferase and control firefly luciferaseactivities were measured using Dual-Glo Luciferase Assay System(Promega, Cat # E2940). Renilla luciferase activity of each well wasdivided by firefly luciferase activity from the same cell to normalizefor different transfection efficiencies across different wells. Thenormalized luciferase activities produced by the Mstn siRNAs werefurther divided by the normalized luciferase activity generated bynon-targeting control siNA to calculate the percent knockdown (% KD) ofreporter expression. All calculations of IC50s were performed usingR.2.9.2 software. The data were analyzed using the sigmoidaldose-response (variable slope) equation for simple ligand binding.

Results—

Four unique, unconjugated siRNAs that target mouse Mstn mRNA sequenceand a non-targeting control siRNA were synthesized. The targetnucleotide sequences of the four Mstn siRNAs are set forth in Table 2a(“target sequence”).

TABLE 2a  Mstn target sequences (5′ to 3′), noting theassigned mouse target site number (column 2) andthe sequence identification number (SEQ ID NO.) (column 3). Target SiteTarget Sequence (mouse) SEQ ID NO: AUGGCAAAGAACAAAUAAU 1167 1GGCAAAGAACAAAUAAUAU 1169 2 ACUCCAGAAUAGAAGCCAU 255 3 UUUGGAAGAUGACGAUUAU421 4

TABLE 2bVarious myostatin-related siNA sense (passenger) and antisense (guide) sequences (5′to 3′) corresponding to the selected target site sequences in Table 2a. Antisense sequences arereadily identified as being complementary to the sense sequence shown. The SEQ ID NOs listedin column 2 correspond to the sense sequences listed in column 3. The SEQ ID NOs listed incolumn 5 correspond to the antisense sequences listed in column 4. SEQSEQ Target Site ID Sense (Target) Sequence Antisense Sequence ID (mouse)NO: (5′ to 3′) (5′ to 3′) NO: 1167 1 AUGGCAAAGAACAAAUAAUAUUAUUUGUUCUUUGCCAU 18 1169 2 GGCAAAGAACAAAUAAUAU AUAUUAUUUGUUCUUUGCC 19255 3 ACUCCAGAAUAGAAGCCAU AUGGCUUCUAUUCUGGAGU 20 421 4UUUGGAAGAUGACGAUUAU AUAAUCGUCAUCUUCCAAA 21

The nucleotide sequences of the Mstn siRNAs and the non-targetingcontrol siRNA are indicated in Table 3. The double-stranded siRNAmolecules within Table 3 contain a sense strand (also known as thepassenger strand) and an antisense strand (also known as the guidestrand), wherein each strand is comprised of 21 nucleotides (position 1(5′) to position 21 (3′)) and contains both internal and terminalchemically-modified nucleotides. The name of each siRNA molecule isprovided in column 1 and corresponds to the mouse Mstn mRNA region towhich the molecule is targeted. Column 2 of Table 3, “Strand”, indicateswhether the particular sequence in the indicated row is the sense (S) orantisense (A/S) strand of the duplex. Column 3 of Table 3, “5-position 1nuc,” describes nucleotide position 1 of the sense and antisense strandsof the indicated siRNA molecules, each comprising of a nucleotide with a5′ cap moiety. The chemical structure of each 5′-position 1 nucleotidesis provided in Table 6a, infra. The nucleotide sequence spanningpositions 2-20 for each of the sense and antisense strands of the siRNAmolecules is described in column 4 of Table 3, wherein the individualnucleotides are separated by a semicolon. The chemical structure of eachnucleotide indicated within column 4 is provided for in Table 6b, infra.The 5th column of Table 3, “Nuc position 21-3′,” represents the 3′ mostnucleotide of the sense or antisense strand of the siRNA, eachrepresented by “omeU-iB” or “omeUSup” (for structures, see Table 6c,infra). The SEQ ID NO: for each strand of the siRNA molecules of Table 3(positions 1-21) is provided in column 6. Each siRNA molecule in Table 3has 3′ overhangs consisting of 2 nucleotides at both ends of themolecule.

TABLE 3 Mstn siRNA sequences 5′- Nuc SEQ siRNA positionNucleotide sequence - position 2 to position ID name Strand 1 nucposition 20 21-3′ NO: Mstn: S 6amiL-omeU; fluG; omeG; fluC; omeA; fluA; omeA; omeU-iB 5 1167 iB-fluAfluG; omeA; fluA; omeC; fluA; omeA; fluA; omeU; fluA; omeA; fluU; omeUsA/S vinylP- fluU; omeU; fluA; omeU; fluU; omeU; fluG; omeUSup 6 moeTomeU; fluU; omeC; fluU; omeU; fluU; omeG; fluC; omeC; fluA; omeU; omeUsMstn: S 6amiL- omeG; fluC; omeA; fluA; omeA; fluG; omeU-iB 7 1169iB-fluG omeA; fluA; omeC; fluA; omeA; fluA; omeU;fluA; omeA; fluU; omeA; fluU; omeUs A/S vinylP-fluU; omeA; fluU; omeU; fluA; omeU; fluU; omeUSup 8 moeTomeU; fluG; omeU; fluU; omeC; fluU; omeU; fluU; omeG; fluC; omeC; omeUsMstn: S 6amiL- omeC; fluU; omeC; fluC; omeA; fluG; omeU-iB 9 255 iB-fluAomeA; fluA; omeU; fluA; omeG; fluA; omeA;fluG; omeC; fluC; omeA; fluU; omeUs A/S vinylP-fluU; omeG; fluG; omeC; fluU; omeU; fluC; omeUSup 10 moeTomeU; fluA; omeU; fluU; omeC; fluU; omeG; fluG; omeA; fluG; omeU; omeUsMstn: S 6 amiL- omeU; fluU; omeG; fluG; omeA; fluA; omeU-iB 11 421iB-fluU omeG; fluA; omeU; fluG; omeA; fluC; omeG;fluA; omeU; fluU; omeA; fluU; omeUs A/S vinylP-fluU; omeA; fluA; omeU; fluC; omeG; fluU; omeUSup 12 moeTomeC; fluA; omeU; fluC; omeU; fluU; omeC; fluC; omeA; fluA; omeA; omeUsPlace- S 6 amiL- omeU; fluC; omeG; fluC; omeC; fluU; omeU-iB 13 bo: 5iB-fluG omeU; fluA; omeU; fluA; omeU; fluC; omeG;fluG; omeU; fluC; omeG; fluA; omeUs A/S vinylP-fluC; omeG; fluA; omeC; fluC; omeG; fluA; omeUSup 14 moeTomeU; fluA; omeU; fluA; omeA; fluG; omeG; fluC; omeG; fluA; omeC; omeUs

Each siRNA (10 nM) was co-transfected along with a Mstn luciferasereporter plasmid (0.6 ng/μl) into cells, and luciferase activity wasmeasured after 48 hours as a reflection of mRNA knockdown (KD). The foursiRNAs show a minimum of 90% Mstn mRNA knockdown and 1050 values lessthan 0.016 nM (see Table 4).

TABLE 4 Mstn siRNA in vitro knockdown activity siRNA Max mRNA KD (%)IC50 (nM) Mstn: 1167 91 0.007 Mstn: 1169 90 0.005 Mstn: 255 94 0.004Mstn: 421 95 0.016

Example 2 Mtsn siRNA Cholesterol Conjugates

Cholesterol Conjugation—

A single cholesterol entity was attached to the 3′ end of the sensestrand of each siRNA molecule described in Table 3. For the preparationof oligonucleotides with a cholesterol unit on the 3′-terminus,deoxycytidylyl-deoxyguanosine (CpG) (see Table 6d, infra, for structure)with a preloaded cholesterol succinate, which also contained adimethoxytrityl (DMT) protected primary alcohol, was used for synthesisof the corresponding oligonucleotide sequence (see Example 1).Typically, the final 5′-DMT protecting group was removed duringoligosynthesis. The oligonucleotide was then cleaved from the CpG bytreatment with a basic solution, such as aqueous methylamine, andpurified by chromatography with a reversed phase resin, such as C18.This purified oligo was annealed to its corresponding complementarystrand to prepare the desired oligonucleotide duplex.

Animals—

Female CD-1 mice were obtained from Charles River and were between 8-9weeks old at time of study. Mice were maintained on a 12-hour light anddark cycle with al libitum access to water and standard chow diet (no.5001; LabDiet). Control and experimental cholesterol-conjugated siRNAswere administered to mice by tail vein injections at indicated dosages.All animal studies were conducted at Merck Research Laboratories in anAAALAC-accredited West Point, Pa. animal facility using protocolsapproved by the Institutional Animal Care and Use Committee (IACUC).

Quantitative Real-Time PCR Analysis—

Mice were sacrificed and tissues were homogenized in Trizol(Invitrogen), extracted in 1-bromo-2-chloropropane (Acros Organics), andtotal RNA was isolated using the MagMax RNA isolation method (Ambion).RNA (125 ng) was reverse transcribed using the High Capacity cDNAReverse Transcription kit (Applied Biosystems, Cat #4368813). TaqmanqPCR analysis was performed using an ABI 7900 Real-Time PCR System usingTaqMan Fast Advanced Master Mix (Applied Biosystems, Cat#4444555). AllTaqman probes and primers were purchased from Applied Biosystems aspre-validated sets: mouse PPIB Assay ID Mm00478295_ml; mouse Mstn AssayID Mm01254559_ml. Taqman data analysis was performed on an ABI 7900Real-Time PCR System, as described previously (Tadin-Strapps, M. et al.,2011, J. Lipid Res. 52:1084-1097).

Serum Analysis of Myostatin Protein—

Blood was collected by tail vein collection, and serum was collected atthe specified time points and analyzed using the GDF-8/MyostatinQuantikine ELISA kit (R&D, Cat # DGDF80). Briefly, serum samples wereactivated as described in manufacturer protocol, with the exception thatthe final activated serum sample had an additional 1:2 dilution incalibrator diluent before assaying.

Results—

In vivo efficacy studies in CD-1 mice were performed using the four MstnsiRNA molecules of Table 3, each of which is conjugated to a singlecholesterol moiety at the 3′ most nucleotide of the sense (passenger)strand. Mice were injected intravenously with 15 mpk siRNA, and MstnmRNA knockdown was assessed in the gastrocnemius muscle after 72 hours(FIGS. 1A and 1B). Reduction of Mstn expression was seen, especiallywith Mstn:1169 and Mstn:1167 cholesterol conjugates. The Mstn:1169cholesterol conjugate was selected for use in follow-up siRNAoptimization and functional studies.

In order to examine the potency and efficacy of theMstn:1169-cholesterol conjugate, mice were treated with a singleinjection of the conjugate at 5, 15, and 50 mpk. Mstn mRNA andcirculating protein levels were measured at various time points (FIG.2). Mstn:1169-cholesterol treatment caused a dose-dependent knockdown inMstn mRNA in gastrocnemius (FIG. 2A), tricep (FIG. 2B) and EDL (FIG. 2C)muscles, demonstrating greater than 8-fold difference in potency betweenthe 5 mpk and 50 mpk doses. Maximum knockdown (KD) was observed with the50 mpk dose at day 7, showing 90-95% mRNA knockdown in all three musclesand 75% knockdown of circulating protein levels (FIG. 2D). Mstn genesilencing with 50 mpk siRNA was between 88-95% KD in gastrocnemius,triceps and spinotrapezius muscles (data not shown), which are mixedfiber type muscles, as well as in the EDL muscle, a predominantly typeII fiber muscle. Mstn mRNA knockdown effects could not be determined inthe soleus muscle due to low Mstn mRNA levels in this muscle (data notshown). Mstn siRNA also displays an extended duration of silencing,maintaining 90-95% mRNA KD and 65% serum Mstn protein levels for 21 daysafter 50 mpk dosing. There was no evidence of liver toxicity and muscledamage at all doses examined, as determined by monitoring serum ALT/ASTand muscle creatine phosphokinase, respectively (data not shown).

Example 3 Prolonged Mstn Knockdown Increases Muscle Size and AltersMuscle Fatigue Profile

Body Composition—

Animal body composition was measured by quantitative magnetic resonancespectroscopy using an EchoMRI instrument (Echo Medical Systems, Houston,Tex.), in order to determine lean/fat mass. Measurements were made 20days after initiation of siRNA dosing.

Micro-CT Imaging and Data Analysis—

Using the LaTheta micro-CT (LaTheta LCT-100A™, Aloka, Echo MedicalSystems, Houston, Tex.), a stack of 10 slices was scanned between theknee and fibula-tibia junction. Images were analyzed as previouslydescribed (Weber, H. et al., 2012, J. Appl. Physiol. 112:2087-98) tofind the largest cross sectional area (CSA) of whole muscle in the lowerleg. Mice were scanned at day-1 with respect to siRNA dosing, and alsoscanned at day 3, 7, 14 and 21.

In Situ Muscle Function Assay—

A custom build in situ assay system, as previously described (see Weber,H. et al., supra), was performed 21 days after siRNA dosing. Briefly,the Achilles tendon of an anesthetized animal was connected to the leverof a combined servomotor/force transducer unit. Electric impulses weredelivered via the sciatic nerve to stimulate the muscles of the lowerleg to contract, while the resulting force was recorded. Tetanicstimulation trains of 50 ms length, containing 4 mA square pulses of 0.1ms duration at 100 Hz, were repeated at a frequency of 0.8 Hz for 300 s.A constant baseline tension of 0.1 N was reestablished betweenstimulations. After completion of the in situ assay, the hind limbmuscles were collected for weighing and histology.

Several parameters representing the fatigue envelopes were extractedfrom a double sigmoidal curve fit, including maximum force (F_(max)),intermediate force (F_(o)), minimum force (F_(min)), early fatigue force(F_(max)-F_(o)), late fatigue force (F_(o)-F_(min)), maximal slope ofearly fatigue (S₁) and late fatigue (S₂) and time constants of early(τ₁) and late fatigue (τ₂) forces.

Measurement of Tibia Bone Lengths—

After sacrificing mice, each hindlimb was stored in 70% ethanolovernight. Next day, legs were stripped of muscle and tissue and lengthwas measured using a digital caliper.

Quantitation of Myofiber Size and Number—

In order to detect muscle fiber cell size, laminin staining wasperformed on gastrocnemius muscle 21 days-post siRNA/control treatment.A transverse cross-section made through the widest region of thegastrocnemius muscle was fixed in 10% formalin overnight. Muscle wasparaffin-embedded and sectioned (5 μm) by standard methods. Sectionsunderwent heat-induced antigen retrieval using citrate buffer (EZAR1,BioGenx #HJ521-XAK) at 103° C. for 10 minutes using a BioGenexEZ-Retriever Microwave. After PBS washes, sections were incubated atroom temperature for 1 hour with a polyclonal Rabbit anti-Lamininantibody at a 1:100 dilution (Abcam, cat # ab11575). A goat anti-rabbitA555 immunofluorescent secondary antibody was applied after further PBSwashes (Invitrogen #A21429), and the slides were mounted usingProlongGold with Dapi (Invitrogen #P36935). Images were captured on aBX-63 Olympus Microscope using a Hamamatsu ORCA-R2 camera and cellSensDimension 1.7 software (Olympus). Muscle fiber area and total number offibers were quantitated on the muscle cross-section using Definienssoftware. Regions out of focus were eliminated from analysis prior tothe segmentation of the image. A multi-resolution algorithm was used toextract the muscle fiber and the endomysiums in the remaining musclesection. Fiber area was determined from 12 mice per group, with1200-7000 cell counts/mouse. Mean fiber size and size frequencydistribution of muscle fibers is shown.

Results—

Since Mstn is known to have a major inhibitory role in muscle growth,the effects of Mstn silencing on muscle mass and function was examined.In order to achieve maximum Mstn knockdown, mice were treated with asingle i.v. 50 mpk dose of the Mstn:1169-siRNA-cholesterol conjugate(see Examples 1 and 2, supra) or controls. After 21 days, mice treatedwith the Mstn siRNA conjugate showed 85-90% Mstn mRNA knockdown ingastrocnemius, EDL, quadriceps, triceps and spinotrapezius muscles (FIG.3A). In addition, circulating levels of Mstn protein were sustainedat >65% reduction through day 21 (FIG. 3B). Muscle size was monitored inboth hind limbs of mice by micro-CT at 3, 7, 14 and 20 days afterdosing. Maximum muscle cross-sectional area (CSA) for each leg,representative of plantarflexor muscle group, was determined byquantitating a series of 10 slices using a custom MATLAB and DefiniensArchitect XD software algorithm. Quantitation results indicate asignificant increase in muscle size by systemic Mstn siRNA-cholesteroltreatment as early as 3 days after initiation of dosing and up to a 20%increase in leg muscle size by day 21 (FIG. 3C). This data is supportedby gastrocnemius muscle weights at day 21/22, which also show ˜20%increase in the weights of both rested and exercised gastrocnemiusmuscle by Mstn-chol treatment in comparison to control mice (FIG. 3D)Laminin immunofluorescent staining of a cross-section of gastrocnemiusmuscle showed that the average fiber cross-sectional area was increased,while the total number of fibers was unaffected by Mstn-chol treatment(FIG. 3E, mean fiber area; FIG. 3F, mean fiber number; FIG. 3G, sizefrequency distribution). In addition to the significant increase inmuscle size observed in mice treated with the Mstn siRNA-cholesterolconjugate, body weight measurements also indicate approximately 10%increase by day 20 (FIG. 311). Body composition analysis, as determinedby quantitative NMR, reveals that this increase in body weight isattributed to an increase in lean mass (FIG. 3I).

In order to assess potential changes to the strength and fiber typecomposition of skeletal muscle as a result of siRNA-mediated genesilencing of Mstn, muscle fatigue response to exercise was assayed in anin situ muscle function assay where repeated isometric contractions areinduced by electrostimulation. Since skeletal muscle consists ofdifferent fiber types, which exhibit different contractile propertiesand differential energy source usage, changes in muscle performance canbe determined by changes in several functional parameters in musclefatigue curves, as described previously (Weber, H. et al., 2012, supra).Briefly, fatigue curves exhibit three stages of muscle fatigue: earlyfatigue, late fatigue and a non-fatigable stage (FIG. 4). “Earlyfatigue” is represented by F_(max)-F₀, indicative of type IIb fibers,which are strong, fast, fatigue very quickly and use creatine phosphateas an energy source. This stage is followed by “late fatigue”(F₀-F_(min)), primarily indicative of type IIa/x fibers, which arestrong, fast, more fatigue-resistant and use glycogen as an energysource. The final stage of the fatigue curve is the “non-fatigable”stage (F_(min)), which is marked type I fibers, which are weak, slow,non-fatigable and use fatty acids as an energy source.

Mstn knockdown results in increased F_(max) and F₀, but since F_(max)-F₀remains unchanged, the “early fatigue” stage is unaffected (FIG. 5A).While there is no significant change in F_(min), F₀-F_(min) isincreased, suggesting an increase in “late fatigue” indicative ofincreased contractile force. This data suggests that Mstn knockdownresults in a change in type IIa/x fibers, due to a potential increase inthe size, quantity and/or strength of the fibers. In addition, there isno change in any of the parameters associated with the timing of thefatigue stages (τ₁, τ₂, S₁, S₂), suggesting that fuel availability andusage are unaffected.

In order to determine changes to muscle quality, “specific force” wascalculated by normalizing muscle contractile force to cross-sectionalarea (CSA) (FIG. 5B). There was no significant difference in specificforce or any additional functional parameter in response to Mstnknockdown, suggesting that there is no effect on muscle quality.

In addition to skeletal muscle, heart has also been reported to expressMstn, although to a lesser degree (Sharma M. et al., 1999, J. CellPhysiol. 180:1-9). Therefore, the heart was examined for potential Mstnknockdown. Quantitative PCR indicates very low Mstn mRNA expression inthe heart (Ct values, 32-35, data not shown) and, therefore, mRNAknockdown could not be determined. There are many reports of changes inheart size in rodent Mstn knockout models or in response to Mstninhibition by small molecule or neutralizing antibodies, althoughobservations are varied, with some reports of cardiac hypertrophy, andother reports of unchanged heart size (Artaza, J. N. et al., 2007, J.Endocrinol. 194:63-76; Morissette, M. R., 2006, Circ. Res. 99:15-24;Rodgers, B. D. et al., 2009, J. Physiol. 587:4873-86; Whittemore, L. A.et al., 2003, Biochem. Biophys. Res. Commun. 300:965-71). In order toaccess signs of cardiac hypertrophy after 21 days of Mstn knockdown,hearts were weighed and normalized to a variety of parameters (FIG. 6).Mstn knockdown resulted in a significant increase in heart weight. Whenheart weight is normalized to tibia length, a parameter anticipated toremain unchanged, mice treated with Mstn siRNA-cholesterol continue todisplay a significant increase in heart weight. However, if heart weightis normalized to body weight (BW) or lean mass, cardiac hypertrophy isno longer observed, suggesting that the increase in heart size iscompensatory to the increased BW resulting from Mstn knockdown.

Example 4 Mechanism of Uptake of Cholesterol Conjugates in Muscle

siRNA Synthesis—

siRNA was synthesized as described in Example 1, supra. The sequences oftwo Ctnnb1 siRNAs used in this Example are indicated in Table 4 (5′-3′direction). A non-targeting control siRNA was also used (see “Placebo 5”from Examples 1 and 2, supra). The content of each column is the same asthose provided in Table 3, supra.

TABLE 5 Ctnnb1 siRNA sequences 5′- Nuc SEQ siRNA positionNucleotide sequence -position 2 to position ID name Strand 1 nucposition 20 21-3′ NO: Ctnnb1: S 6amiL-omeU; fluG; omeU; omeU; fluG; fluG; fluA; omeU- 15 1797 iB-omeU; omeU; fluG; fluA; omeU; omeU; iBSup [3′ Chol] omeComeC; fluG; omeA; fluA; fluA; omeUs A/S vinylP-fluU; omeU; fluC; omeG; fluA; omeA; fluU; omeUSup 16 moeTomeC; fluA; omeA; fluU; omeC; fluC; omeA; fluA; omeC; fluA; omeG; omeUsCtnnb1: S iB- omeU; fluG; omeU; omeU; fluG; fluG; fluA; omeU- 17 1797omeC omeU; omeU; fluG; fluA; omeU; omeU; iBSup [5′ Chol]omeC; fluG; fluA; fluA; fluA; omeUs A/S vinylP-fluU; omeU; fluC; omeG; fluA; omeA; fluU; omeUSup 16 moeTomeC; fluA; omeA; fluU; omeC; fluC; omeA; fluA; omeC; fluA; omeG; omeUs

Animals—

Female C57BL/6J wild-type LDL receptor −/− and ApoE −/− mice wereobtained from Jackson Laboratory (Stock #002207, 002052, respectively)and were between 25-27 weeks old at time of study. Female CD-1 mice wereobtained from Charles River and were between 8-9 weeks old at time ofstudy. All mice were maintained on a 12-hour light and dark cycle withal libitum access to water and standard chow diet (no. 5001; LabDiet).Control and experimental cholesterol-conjugated siRNAs were administeredto mice by tail vein injections at indicated dosages. Blood wascollected by cardiac puncture at the time of harvest. Tissue sampleswere collected at specific time points after dosing. All animal studieswere conducted at Merck Research Laboratories in an AAALAC-accreditedWest Point, Pa. animal facility using protocols approved by theInstitutional Animal Care and Use Committee (IACUC).

Results—

In order to examine the potential mechanism of uptake of cholesterolconjugates in muscle and whether association of the conjugate withlipoprotein particles is required for uptake, the efficacy of Ctnnb1siRNA molecules with a single cholesterol entity attached to either the3′ or 5′ end of the passenger strand was examined in wildtype, LDLreceptor (LDLR) −/− and ApoE −/− mice. Mice were treated with a singlei.v. injection of Ctnnb1:1797[3′ Chol] siRNA at 14 mpk and Ctnnb1 mRNAwas measured in the gastrocnemius muscle after 72 hours (FIG. 7A). Amaximum of 60% mRNA knockdown was observed in wt mice. There is areduction in mRNA KD in ApoE −/− mice (50% KD) and a trend towardreduced KD in LDLR −/− mice. The data suggests that Ctnnb1 cholesterolconjugate uptake is only partially mediated through the LDL receptor orvia any ApoE-containing lipoprotein in muscle.

In order to determine whether the position of the cholesterol on thepassenger strand is essential for cholesterol conjugate efficacy,Ctnnb1-chol conjugates with cholesterol on either the 3′ or 5′ end ofthe passenger strand were examined in vivo (FIG. 7B). CD-1 mice weretreated with 15 mpk of either version of Ctnnb1-chol and compared to aPBS control and a Placebo 5-chol, which has cholesterol at the 3′position. Ctnnb1 mRNA levels were examined in gastrocnemius muscle 72hours after injection. The data suggests that both positions can be usedto create highly efficacious cholesterol conjugates in muscle.

Example 5 Chemical Structures of the Chemically-Modified NucleotidesUsed to Generate the siRNA Molecules Exemplified Herein

TABLE 6a Structure of 5′-position 1 nucleotides contained within siRNAmolecules in Tables 3 and 5. 5′-position 1 nuc Structure vinylPmoeT

6amil-iB-fluX X = B = Base U, G, C, A

6amil-iB-omeX X = B = Base U, G, C, A

TABLE 6b Structure of internally-located nucleotides (i.e., positions2-20) contained within the siRNA molecules in Tables 3 and 5. Internalnuc (positions 2-20) Structure omeX X = B = Base U, G, C, A

omeXs X = B = Base U, G, C, A

fluX X = B = Base U, G, C, A

fluXs X = B = Base U, G, C, A

TABLE 6c Structure of nucleotide position 21-3′ nucleotides exemplifiedin Tables 3 and 5. Nuc position 21-3′ Structure omeU-Sup

omeU-iBSup

TABLE 6d Structure of cholesterol CpG Name Structure Choles- terol CpG

1. A method of modulating in vivo expression of a myostatin gene in asubject comprising introducing to said subject by systemicadministration an effective amount of a myostatin siNA conjugate, or apharmaceutical composition comprising said siNA conjugate, wherein thesiNA conjugate comprises an siNA molecule that targets a myostatin geneexpressed by said subject linked to a lipophilic moiety, and wherein thesiNA conjugate mediates RNA interference.
 2. A method according to claim1, wherein the lipophilic moiety is cholesterol.
 3. A method accordingto claim 1, wherein the lipophilic moiety is attached to a 3′-end of thesiNA molecule.
 4. A method according to claim 1, wherein the siNAmolecule comprises one or more chemically-modified nucleotides.
 5. Amethod according to claim 1, wherein the siNA molecule is adouble-stranded molecule comprising an antisense strand and a sensestrand, wherein said antisense strand is complementary to said sensestrand.
 6. A method according to claim 5, wherein the antisense strandand the sense strand are each independently 15 to 30 nucleotides inlength.
 7. A method according to claim 5, wherein the siNA moleculecomprises one or more 3′-overhanging nucleotides on one or both strands.8. A method according to claim 5, wherein the lipophilic moiety isattached to either the 3′-end of the sense strand of the siNA molecule,the 5′-end of the sense strand of the siNA molecule, or the 3′-end ofthe antisense strand of the siNA molecule.
 9. A method according toclaim 1, wherein the siNA molecule comprises a cap on a 3′-end of themolecule.
 10. A method according to claim 1, wherein the subject is ahuman.
 11. A method according to claim 1, wherein the subject islivestock.
 12. A method of enhancing muscle mass in a subject comprisingreducing myostatin levels in said subject by introducing to said subjectby systemic administration an effective amount of a myostatin siNAconjugate, or a pharmaceutical composition comprising said siNAconjugate, wherein the siNA conjugate comprises an siNA molecule thattargets a myostatin gene expressed by said subject linked to alipophilic moiety, and wherein the siNA conjugate mediates RNAinterference.
 13. A method of enhancing muscle performance mass in asubject comprising reducing myostatin levels in said subject byintroducing to said subject by systemic administration an effectiveamount of a myostatin siNA conjugate, or a pharmaceutical compositioncomprising said siNA conjugate, wherein the siNA conjugate comprises ansiNA molecule that targets a myostatin gene expressed by said subjectlinked to a lipophilic moiety, and wherein the siNA conjugate mediatesRNA interference.
 14. A method of treating a musculoskeletal disease ordisorder, or a disease or disorder that results in conditions in whichmuscle is adversely affected, in a subject comprising reducing myostatinlevels in said subject by introducing to said subject by systemicadministration an effective amount of a myostatin siNA conjugate, or apharmaceutical composition comprising said siNA conjugate, wherein thesiNA conjugate comprises an siNA molecule that targets a myostatin geneexpressed by said subject linked to a lipophilic moiety, and wherein thesiNA conjugate mediates RNA interference. 15-18. (canceled)
 19. Adouble-stranded short interfering nucleic acid (siNA) molecule thatinhibits the expression of myostatin, wherein: (a) the siNA comprises asense strand and an antisense strand; (b) each strand is independently15 to 30 nucleotides in length; and, (c) the antisense strand comprisesat least 15 nucleotides having sequence complementary to any of:(SEQ ID NO: 1) 5′- AUGGCAAAGAACAAAUAAU -3′; (SEQ ID NO: 2)5′- GGCAAAGAACAAAUAAUAU -3′; (SEQ ID NO: 3) 5′- ACUCCAGAAUAGAAGCCAU -3′;or (SEQ ID NO: 4) 5′- UUUGGAAGAUGACGAUUAU -3′.


20. A double-stranded short interfering nucleic acid (siNA) moleculethat inhibits the expression of myostatin, wherein: (a) the siNAcomprises a sense strand and an antisense strand; (b) each strand isindependently 15 to 30 nucleotides in length; and (c) the antisensestrand comprises at least a 15 nucleotide sequence of: (SEQ ID NO: 18)5′- AUUAUUUGUUCUUUGCCAU -3′; (SEQ ID NO: 19)5′- AUAUUAUUUGUUCUUUGCC -3′; (SEQ ID NO: 20)5′- AUGGCUUCUAUUCUGGAGU -3′; or (SEQ ID NO: 21)5′- AUAAUCGUCAUCUUCCAAA -3′;

and wherein one or more of the nucleotides are optionally chemicallymodified.
 21. A double-stranded siNA molecule of claim 20, wherein thesiNA molecule comprises any of: (SEQ ID NO: 1)5′- AUGGCAAAGAACAAAUAAU -3′ and (SEQ ID NO: 18)5′- AUUAUUUGUUCUUUGCCAU -3′; (SEQ ID NO: 2) 5′- GGCAAAGAACAAAUAAUAU -3′and (SEQ ID NO: 19) 5′- AUAUUAUUUGUUCUUUGCC -3′; (SEQ ID NO: 3)5′- ACUCCAGAAUAGAAGCCAU -3′ and (SEQ ID NO: 20)5′- AUGGCUUCUAUUCUGGAGU -3′; or (SEQ IN NO: 4)5′- UUUGGAAGAUGACGAUUAU -3′ and (SEQ ID NO: 21)5′- AUAAUCGUCAUCUUCCAAA -3′.


22. The double-stranded siNA molecule of claim 19, wherein the siNAmolecule is linked to a lipophilic moiety.
 23. The double-stranded siNAmolecule of claim 22, wherein the lipophilic moiety is cholesterol. 24.The double-stranded siNA molecule of claim 23, wherein the lipophilicmoiety is attached to a 3′-end of the siNA molecule.
 25. Thedouble-stranded siNA molecule of claim 23, wherein the lipophilic moietyis attached to a 5′-end of the siNA molecule.